Methods and compositions for modulating drug activity through telomere damage

ABSTRACT

The invention provides methods and compositions for modulating the activity of therapeutic agents for the treatment of a cancer by administering one or more agents that (either alone or in combination) induces telomere damage and inhibits telomerase activity in the cancer cell. The method initially uses, e.g., a telomere damage-inducing agent such as paclitaxel, and a telomerase inhibitory agent such as AZT. The invention also provides methods for identifying other agents with telomere damage-inducing activity and/or telomerase inhibitory activity (as well as and compositions having such activity), for use in the treatment of cancer.

RELATED INFORMATION

This application claims priority to U.S. Provisional Application Ser.No. 60/137,549, entitled “Method to Enhance Taxol Efficacy and TreatmentEffects,” filed on Jun. 4, 1999, the entire contents of which are herebyincorporated herein by reference. The contents of all patents, patentapplications, and references cited throughout this specification arehereby incorporated by reference in their entireties.

GOVERNMENT SPONSORED RESEARCH

This work was supported, in part, by grants from the United StatesDepartment of Health and Human Services.

BACKGROUND OF THE INVENTION

Telomeres are the structures capping the ends of chromosomes, and arecritical to the maintenance of chromosomal integrity and replicationpotential. Telomere length is lost during each cycle of proliferation,and reduction below a critical minimum length results in cell death(Lingner, J., et al. (1995) Science 269:1533-1534). The enzymetelomerase is capable of restoring telomere length, and is nearlyuniversally present in tumor cells, while usually absent in normal cells(Hiyama, E., et al. (1996) J. Natl. Cancer Inst., 88:116-122). Thecritical nature of its function, and its selective presence in tumorcells, suggest it is a desirable target for cancer chemotherapy.Unfortunately, however, telomerase inhibitors do not appear to havesignificant antitumor activity, probably because telomere shorteningoccurs slowly, thereby allowing more cycles of undesired cellproliferation. In addition, current techniques for measuring changes intelomerase activity and telomere biology are of limited sensitivity.

Accordingly, a need exists for developing more efficacious cancertherapies, including combination drug therapies, that specificallytarget cellular events associated with the cancer phenotype. Inaddition, improved techniques for measuring cellular changes associatedwith the cancer phenotype, e.g., telomere biology, are needed.

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the discovery thatpaclitaxel treatment of a cancer causes telomere damage thereby inducingtelomerase activity and leading to resistance to paclitaxel treatment inthe cancer. By treating the cancer with paclitaxel and a telomeraseinhibitory agent, such as AZT, telomere damage induced resistance topaclitaxel is avoided.

Accordingly, the present invention provides for the efficient discoveryof new cancer therapies and the enhancement of existing cancer therapiesby recognizing that a desired anti-cancer agent advantageously has twoactivities: 1) telomere damage-inducing activity; and 2) telomeraseinhibitory activity. Thus, the invention provides methods forintelligently exploiting drug interactions and the cellular mechanismthey target (e.g., telomere maintenance), to achieve better methods andcompositions for the treatment of abnormal cellular growth, e.g.,cancer.

The present invention also provides superior treatment methods andcompositions that surpass existing ones that rely on the targeting of,e.g., only one cellular event such as, e.g., telomerase activity orimpairment of DNA replication. This is especially advantageous in thetreatment of a complex and highly diverse disease such as cancer whichfrequently comprises cells having multiple genetic alterations. Stillfurther, the invention provides improved techniques for measuringchanges in telomere biology.

Accordingly, the present invention provides several advantages overcurrent methods and compositions for treating a cancer, that include,but are not limited to, the following:

providing a method for treating a cancer by inducing telomere damage inthe cancer cell, and concurrently blocking telomerase activity to repairthereby the damaged telomeres;

providing a screening method for identifying an agent or agents capableof inducing telomere damage and/or telomerase inhibitory activity toprovide thereby potent anti-cancer agents;

providing a method of identifying anticancer synergy between agents thattogether exhibit therapeutically effective telomere damage-inducingactivity and telomerase inhibitory activity (which allows for theability to administer less toxic doses to the patient);

providing an pharmaceutical composition suitable for use as ananticancer therapy and having a telomere damage-inducing activity aswell as a telomerase inhibitory activity;

providing a method for enhancing the efficacy of an anticancer agent bycoadministering a telomerase inhibitory agent;

providing various compositions of agents having a telomeredamage-inducing activity and telomerase inhibitory activity and furtherformulated for timed-release or specific targeting of a cancer cell ortissue; and

improved methods of increased sensitivity for measuring telomeraseactivity.

Accordingly, in one aspect, the present invention provides a method forinhibiting or reducing the growth of a cell by administering a dose of atelomere damage-inducing agent to the cell, and administering a dose ofa telomerase inhibitory agent to the cell, such that an inhibition orreduction in the growth of the cell is achieved.

In a related second aspect, the invention provides a method forinhibiting or reducing the growth of a cell by obtaining an agentselected from the group consisting of a telomere damage-inducing agentand a telomerase inhibitory agent, administering a dose of a telomeredamage-inducing agent to the cell and administering a dose of atelomerase inhibitory agent to the cell, such that an inhibition orreduction in the growth of the cell is achieved.

In one embodiment of the above related aspects, the method may be usedwhere the growth of a cell is aberrant, preferably, e.g., a tumor cellor a leukemic cell. The tumor cell may be either benign, premalignant,or malignant and/or be characterized by either hyperplastic andhypertrophic growth. Still further, the cell may be in a human ortreated ex vivo. Moreover, the tumor cell may be of any cell type suchas, e.g., occur in the brain, breast, ovary, testes, bladder, prostate,colon, lung, liver, pancreas, or uterus. Similarly, the leukemic cellmay be of any cell type known to occur in the blood such as, e.g., anerythrocyte (i.e., an erythroleukemia), myelocyte (i.e., a myeloidleukemia), or lymphocyte (i.e., a leukemia or lymphoma).

In another embodiment, the inhibition or reduction in the growth of thecell includes apoptosis (i.e., programmed cell death) or necrosis.

In yet another embodiment, the telomere damage-inducing agent andtelomerase inhibitory agent are administered serially (e.g., in eitherorder), or preferably, concurrently, and may be administered as, e.g., atimed-release formulation. Still further, the telomere damage-inducingagent and/or telomerase inhibitory agent may be administered locally,regionally, or preferably, systemically.

In even another embodiment, the telomere damage-inducing agent ispaclitaxel, or a derivative thereof, and the telomerase inhibitory agentis a nucleotide analog, or derivative thereof, and in a preferredembodiment, AZT or d4T. In a related embodiment, the telomeraseinhibitory agent is an antisense nucleic acid corresponding to atelomerase.

In still another embodiment, the telomere damage-inducing agent and/ortelomerase inhibitory agent, is administered at a subtherapeutic dose.

In a third aspect, the invention provides a method of identifying anagent capable of inhibiting or reducing the growth of a cell bycontacting a cell with at least one agent and determining if telomeredamage has occurred, whereby an agent determined to be capable ofinducing telomere damage, is indicated as capable of inhibiting orreducing the growth of a cell.

In a related fourth aspect, the invention provides a method ofidentifying an agent capable of inhibiting or reducing the growth of acell by contacting a cell with at least one agent, and determining iftelomere damage has occurred, contacting a cell with the same or atleast one other agent, and determining if a reduction in telomeraseactivity has occurred, whereby an agent or agents, alone or incombination, that are determined to be capable of inducing telomeredamage and reducing telomerase activity, are indicated as capable ofinhibiting or reducing the growth of a cell.

In one embodiment of the above aspects, the invention encompasses anagent or agents identified according to the method of the foregoingaspects. In a related embodiment, the agent or agents are preferably apharmaceutical composition containing such agent or agents incombination with a pharmaceutically acceptable carrier. In anotherrelated embodiment, the invention provides a method of inhibiting orreducing the growth of a cell preferably, aberrant cell growth in amammal, e.g., a human, by administering to a cell, a therapeuticallyeffective amount of an agent or agents identified according to themethod of the foregoing aspects.

In a fifth aspect, the invention provides a composition suitable forinhibiting or reducing the growth of a cell containing an agent having atherapeutically effective amount of telomere damage-inducing activityand an agent having a therapeutically effective amount of telomeraseinhibitory activity.

In a sixth aspect, the invention provides an article of manufacturecontaining a vial containing a purified telomere damage-inducing agentand a purified telomerase inhibitory agent, and instructions for use.

In one embodiment of the foregoing aspect, the purified telomeredamage-inducing agent and purified telomerase inhibitory agent arepackaged in separate vials.

In a seventh aspect, the invention provides a method of treating acancer in a patient by identifying a patient having or about to have acancer, administering a telomere damage-inducing agent to the patient,and administering a telomerase inhibitory agent to the patient such thattreatment of the cancer is achieved.

In a related eighth aspect, the invention provides a method of treatingcancer in a patient by obtaining an agent selected from the groupconsisting of a telomere damage-inducing agent and a telomeraseinhibitory agent, identifying a patient having or about to have acancer, administering a telomere damage-inducing agent to the patient,and administering a telomerase inhibitory agent to the patient such thattreatment of the cancer is achieved.

In one embodiment of either of the foregoing aspects, the telomeredamage-inducing agent is paclitaxel, or a derivative thereof, and thetelomerase inhibitory agent is a nucleotide analog, or derivativethereof, preferably AZT. In a related embodiment, the telomeraseinhibitory agent is an antisense nucleic acid corresponding to atelomerase.

In a ninth aspect, the invention provides a method of enhancing theefficacy of an anticancer agent by administering the agent in thepresence of a telomerase inhibitory agent, whereby the efficacy of theanticancer agent is increased as compared to a control.

In one embodiment, the anticancer agent is a telomere damage-inducingagent, preferably paclitaxel.

In another embodiment, the telomerase inhibitory agent is a nucleotideanalog, a derivative thereof, and preferably, AZT.

In still another embodiment, the telomerase inhibitory agent is anantisense nucleic acid corresponding to a telomerase.

In a tenth aspect, the invention provides a method of reducing orinhibiting the resistance of a cell to an anticancer agent byadministering the anticancer agent in the presence of a telomeraseinhibitory agent, whereby the resistance of a cell to an anticanceragent is decreased as compared to a control.

In one embodiment, the agent is a telomerase inhibitory agent, such as anucleotide analog, and preferably AZT or d4T.

In another embodiment, the telomerase inhibitory agent is an antisensenucleic acid corresponding to a telomerase.

In an eleventh aspect, the invention provides a method for detectingtelomerase activity in cell extract by incubating a reaction mixturecomprising a cell extract, a nucleic acid substrate for a telomerase,and nucleotide triphosphates for a time sufficient for the nucleic acidsubstrate to be polymerized; and contacting the substrate with at leastone nucleic acid primer and subjecting the substrate to a polymerasechain reaction; and detecting the presence of polymerase chain reactionproducts to detect thereby telomerase activity in the cell extract.

In one embodiment, the telomerase extract is derived from a cell, thathas been contacted with an agent, or the telomerase extract is contactedwith the agent.

In another embodiment, the agent is a telomerase inhibitory agent, suchas a nucleotide analog, and preferably AZT or d4T.

In a related embodiment, the telomerase inhibitory agent is an antisensenucleic acid corresponding to a telomerase.

In a preferred embodiment, the telomerase extract has been derived froma human cell.

In another embodiment, the nucleic acid substrate comprises the sequenceprovided in SEQ ID NO: 10.

In another embodiment, the nucleic acid primer comprises the sequenceprovided in SEQ ID NOS: 1 and/or 2.

In still another embodiment, the nucleic acid primer can be labeled witheither a radioisotope or fluorescent label.

In a twelfth aspect, the invention provides a method for determiningtelomere length by hybridizing a telomeric DNA fragment with a telomereprobe; and determining the amount of hybridized telomere probe present,whereby the amount of hybridized telomere probe present is an indicationof telomere length.

In one embodiment, the chromosomal nucleic acid fragments are producedusing a restriction enzyme, preferably, for example, HinfI, HaeIII,HhaI, and more preferably a combination thereof.

In another embodiment, the telomeric DNA is derived from a cell,preferably a human cell.

In another embodiment, the cell that has been contacted with an agent,preferably a telomerase inhibitory agent, more preferably a nucleotideanalog, and most preferably, AZT or d4T. In another related embodiment,the telomerase inhibitory agent is an antisense nucleic acidcorresponding to a telomerase.

In yet another embodiment, the telomere probe comprises the sequenceprovided in SEQ ID NO: 10. In a related embodiment, the telomere probemay be labeled with a radioisotope or fluorescent label.

In a thirteenth aspect, the invention provides a method of identifying atelomerase inhibitory agent by contacting a cell with an agent;incubating a reaction mixture comprising an extract of the cell, anucleic acid substrate for a telomerase, and nucleotide triphosphatesfor a time sufficient for the nucleic acid substrate to be polymerized;and contacting the substrate with at least one nucleic acid primer andsubjecting the substrate to a polymerase chain reaction; and detecting adecrease in the presence of polymerase chain reaction products tothereby identify a telomerase inhibitory agent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Transient induction of telomerase by paclitaxel (A) Humanpharynx FaDu cells were treated with 200 nM paclitaxel. (B)Histocultures of human pharynx FaDu xenograft tumors were treated with 1μM paclitaxel. The treatment-induced changes in telomerase activity, asa function of treatment duration, was measured using TRAP. Results arestandardized to the telomerase activity in untreated control cells.

FIG. 2. Presence of DNA free ends induces telomerase. Human pharynx FaDucells were transfected with linearized DNA fragments with free endsunprotected by telomeres. The linearized DNA fragments were labeled withfluorescein-labeled dNTPs. The transfected cells containing the labeledDNA fragments were detected with flow cytometry and separated from thenon-transfected cells by cell sorting. The telomere activity innontransfected and transfected cells was measured using TRAP.Transfected cells: +, nontransfected cells: −.

FIG. 3. AZT enhances the reduction of cell number by paclitaxel: Fixedconcentration method. Human pharynx FaDu cells were treated with AZT at10 or 20 μM for 24 hr, after which varying concentrations of paclitaxelwere added and incubated for an additional 24 hr. The drug effect wasmeasured using the sulforhodamine B assay which determines the totalnumber of cells remained attached to the culture flasks. Results areexpressed as % of the untreated controls.

FIG. 4. AZT enhances the reduction of cell number by paclitaxel: Fixedratio method. Human pharynx FaDu cells were treated with AZT for 24 hr,after with varying concentrations of paclitaxel were added and incubatedfor an additional 24 hr. The concentrations of AZT and paclitaxel weremaintained at fixed ratios (i.e., 80:20, 60:40, 40:60, 20:80) of theirrespective IC₅₀ values (i.e., concentrations required to reduce thetotal cell numbers by 50% when used as single agents). For example, aconstant ratio of AZT to paclitaxel of 80%:20% would represent acombination containing a concentration of AZT equal to the multiples(e.g., 0.25, 0.5, 1, 2, 3 and 4 times) of the 80% value of the IC₅₀ ofAZT with a concentration of paclitaxel equal to the multiples of the 20%value of the IC₅₀ of paclitaxel. For the fixed ratio method, theconcentration range was between 5 to 300 μM for AZT and 2.5 to 160 nMfor paclitaxel. The drug effect was measured using the sulforhodamine Bassay which measures the total number of cells that remained attached tothe culture flasks. Results are expressed as % of the untreatedcontrols.

FIG. 5. Analysis of nature of interaction between AZT and paclitaxel.The nature of interaction between paclitaxel and AZT was analyzed by theisobologram method. Concentration-effect curves generated for both drugsand their combinations, as shown in FIG. 4, were used to determine theconcentration of each compound, either alone or in combination, neededto achieve a given level of effect. A combination index value of 1indicates additive interaction, values less than 1 indicate synergisticaction, and values greater than 1 indicate antagonistic interaction. Thecombination indices for the different combinations of AZT and paclitaxelin different concentration ratios, as described in FIG. 4, at differenteffect levels (i.e., ranging from 10 to 90% of cell number reduction)are shown.

FIG. 6. AZT enhances the cell detachment effect of paclitaxel. Humancancer cells were treated with AZT for 24 hr, after which varyingconcentrations of paclitaxel were added and incubated for an additional48 hr. The AZT concentration was selected such that this concentrationproduced 50% reduction in total cell number, but did not causeappreciable detachment of cells from the growth surface (i.e., <10%) norapoptosis that was significantly higher than in the untreated controls.The selected AZT concentration was 10 μM for FaDu and PC3 cells, 5 μMfor MCF7 cells, and 100 μM for SKOV-3 cells.

FIG. 7. d4T enhances the cell detachment effect of paclitaxel Humanpharynx FaDu cancer cells were treated with 20 or 40 μM d4T for 24 hr,after which varying concentrations of paclitaxel were added andincubated for an additional 48 hr.

FIG. 8. AZT enhances the paclitaxel-induced release of DNA-histonecomplex from the nucleus. Human cancer cells were treated with AZT for24 hr, after which varying concentrations of paclitaxel were added andincubated for an additional 48 hr. The AZT concentration was selectedsuch that this concentration produced 50% reduction in total cellnumber, but did not cause appreciable detachment of cells from thegrowth surface (i.e., <10%) nor apoptosis that was significantly higherthan in the untreated controls. The selected AZT concentration was 10 μMfor FaDu and PC3 cells, 5 μM for MCF7 cells. The treatment-inducedapoptosis was measured using Cell Death Detection ELISA which quantifiesthe amount of DNA-histone complex released into the cytoplasm asabsorbance at 405 nm.

FIG. 9. AZT enhances the tumor growth retardation by paclitaxel. Theantitumor activity of paclitaxel, with or without AZT, was evaluated inimmunodeficient mice bearing the human pharynx FaDu xenografts (>15 mm³in size prior to drug treatment). The four treatment groups are: salinecontrol, AZT, paclitaxel, paclitaxel+AZT. The saline control groupreceived injections of 200 μl/day of physiological saline of fiveconsecutive days. The paclitaxel group received injections of 10mg/kg/day paclitaxel dissolved in Cremophor and ethanol (i.e., Taxol) ina volume of 200 μl for five consecutive days. The AZT group received aseven-day infusion of AZT at a rate of 200 ng/hr by a subcutaneouslyimplanted Alzet minipump. The paclitaxel+AZT group received the combinedtreatment of the paclitaxel group and the AZT group, where the AZTinfusion was started one day prior to the start of the paclitaxelinjections.

FIG. 10. AZT enhances the antitumor activity of paclitaxel in animalsbearing human xenograft tumors: Kaplan Meier analysis. The antitumoractivity of paclitaxel, with or without AZT, was evaluated inimmunodeficient mice (male Balb/c nu/nu mice, 6-8 weeks old) bearing thehuman pharynx FaDu xenografts (>15 mm³ in size prior to drug treatment).The four treatment groups are: saline control, AZT, paclitaxel,paclitaxel+AZT. The saline control group received injections of 200μl/day of physiological saline of five consecutive days. The paclitaxelgroup received injections of 10 mg/kg/day paclitaxel dissolved inCremophor and ethanol (i.e., Taxol) in a volume of 200 μl for fiveconsecutive days. The AZT group received a seven-day infusion of AZT ata rate of 200 ng/hr by a subcutaneously implanted Alzet minipump. Thepaclitaxel+AZT group received the combined treatment of the paclitaxelgroup and the AZT group, where the AZT infusion was started one dayprior to the start of the paclitaxel injections. The ability of drugtreatment to prolong the survival time was measured. The animals weremonitored for 100 days, or until moribundity, as defined by a tumorlength exceeding 1.0 cm, was reached.

DETAILED DESCRIPTION OF THE INVENTION

In order to provide a clear and consistent understanding of theinvention, certain terms employed in the specification, examples, andthe claims are, for convenience, collected here.

DEFINITIONS

As used herein, the term “aberrant growth” refers to a cell phenotypewhich differs from the normal phenotype of the cell, particularly thoseassociated either directly or indirectly with a disease such as cancer.

The term “administering” refers to the introduction of an agent to acell, e.g., in vitro, a cell in an animal, i.e., in vivo, or a celllater placed back in the animal (i.e., ex vivo).

The terms “agent”, “drug”, “compound”, “anticancer agent”,“chemotherapeutic”, and “antitumor agent” are used interchangeably andrefer to agent/s (unless further qualified) that have the property ofinducing telomere damage or possess telomerase inhibitory activity (orboth) and are thus suitable (either alone or in combination) forinhibiting or reducing aberrant cell growth, e.g., a cancer. The term“agent” includes small molecules, macromolecules (e.g., peptides,proteins, antibodies, or antibody fragments), and nucleic acids (e.g.,gene therapy constructs, recombinant viruses, nucleic acid fragments(including, e.g., synthetic nucleic acid fragments). The foregoing termsare also intended to include cytotoxic, cytocidal, or cytostatic agentswhich may be used in combination with a telomere damage-inducing agentand/or telomerase inhibitory agent, or represent candidate agents thathave such properties which can be readily determined using the methodsof the invention. As used herein, the term “an agent” (e.g., “a telomeredamage-inducing agent” or “a telomere inhibitory agent”) is meant toinclude at least one agent, i.e., a single agent or two or more agents.

The term “antisense nucleic acid corresponding to a telomerase” refersto a nucleic acid that can inhibit or reduce telomerase activity by,e.g., interacting with a nucleic acid component of a telomerase enzymecomplex or a nucleic acid transcript encoding a component of thetelomerase.

The term “apoptosis” refers to any non-necrotic, cell-regulated form ofcell death, as defined by criteria well established in the art.

The terms “benign”, “premalignant”, and “malignant” are to be giventheir art recognized meanings.

The terms “cancer”, “tumor cell”, tumor”, “leukemia”, or “leukemic cell”are used interchangeably and refer to any neoplasm (“new growth”), suchas a carcinoma (derived from epithelial cells), adenocarcinoma (derivedfrom glandular tissue), sarcoma (derived from connective tissue),lymphoma (derived from lymph tissue), or cancer of the blood (e.g.,leukemia or erythroleukemia). The term cancer or tumor cell is alsointended to encompass cancerous tissue or a tumor mass which shall beconstrued as a compilation of cancer cells or tumor cells. In addition,the term cancer or tumor cell is intended to encompass cancers or cellsthat may be are either benign, premalignant, or malignant. Typically acancer or tumor cell exhibits various art recognized hallmarks such as,e.g., growth factor independence, lack of cell/cell contact growthinhibition, and/or abnormal karyotype. By contrast, a normal celltypically can only be passaged in culture for a finite number ofpassages and/or exhibits various art recognized hallmarks attributed tonormal cells (e.g., growth factor dependence, contact inhibition,morphological changes, tissue architecture, and/or a normal karyotype).

The term “cell” includes any eukaryotic cell, such as somatic or germline mammalian cells, or cell lines, e.g., HeLa cells (human), NIH3T3cells (murine), embryonic stem cells, and cell types such ashematopoietic stem cells, myoblasts, hepatocytes, lymphocytes, andepithelial cells and, e.g., the cell lines described herein.

The term “derivative” as in “paclitaxel or a derivative thereof” refersto a chemical compound derived from a parent compound (e.g., paclitaxel)that contains the essential elements and/or features of the parentcompound but differs from the parent compound by one or more elements,substituents and/or functional groups such that the derivative has thesame or similar biological properties/activities (e.g., telomeredamage-inducing properties).

The terms “hybridize”, “hybridized”, or “hybridization” are art-knownand refer to the interaction of complementary DNA and/or RNA sequencesto form a duplex molecule. Typically, hybridization takes place betweena primer and template but may also take place between an antisensemolecule and a nucleic acid template that is a transcript or a componentof a nucleic acid and polypeptide complex, such as, e.g., a telomerase.Conditions and degrees of correspondence needed between various nucleicacid templates are well described in the art.

The term “identifying a patient having or about to have cancer” refersto a patient having been determined to have, or to be statisticallylikely to have, a cancer using various art recognized diagnostic orprognostic techniques including, e.g., the PSA test, BRCA1 and/or BRCA2genotyping, genetic profiling, etc. The term is also intended to includethe mere knowing or receipt of any information (e.g., a prognosis,diagnosis) indicating that the patient is having or about to have acancer.

The term “inhibiting or reducing the growth of a cell” e.g., a cancercell, refers to the slowing, interrupting, or arresting of its growthand/or metastasis, and can, but does not necessarily require, e.g., atotal elimination of the aberrant growth of the cell. The term is alsointended to encompass inhibiting or reducing cell growth via cell death(apoptosis) or necrosis.

The terms “locally”, “regionally”, “systemically” refer to,respectively, the administration of a therapy “locally”, e.g., into atumor mass, “regionally”, e.g., in a general tumor field or areasuspected to be seeded with metastases, or “systemically” e.g., orallyor intravenously with the intent that the agent will be widelydisseminated throughout the subject.

The term “nucleic acid primer” includes short single-strandedoligonucleotides that, typically, are between about 10 to 100 bases andare designed to hybridize with a corresponding template nucleic acid.Primer molecules may be complementary to either the sense or theanti-sense strand of a template nucleic acid and are typically used ascomplementary pairs that flank a nucleic acid region of interest and,when appropriately bound to a template, can serve as an origin of nucleiacid polymerization, including, e.g., a polymerase chain reaction.

The term “nucleic acid substrate” refers to the minimal template nucleicacid required by a telomerase in order to initiate a detectablepolymerization of a nucleic acid, at for example, the end of achromosome or an experimental representation thereof, e.g., a syntheticnucleic acid template.

The term “nucleotide analog, or derivative thereof” refers to those artrecognized modified nucleic acid bases that, typically, resemble anatural building block of DNA or RNA polymerization but have beenmodified to have an additional property such as, e.g., the ability toinhibit a reverse transcriptase, e.g., retroviral reverse transcriptasesand telomerases.

The term “pharmaceutically acceptable carrier” is art recognized andincludes a pharmaceutically acceptable material, composition or vehicle,suitable for administering compounds of the present invention tomammals.

The term “pharmaceutical composition” includes preparations suitable foradministration to mammals, e.g., humans. When the compounds of thepresent invention are administered as pharmaceuticals to mammals, e.g.,humans, they can be given per se or as a pharmaceutical compositioncontaining, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) ofactive ingredient (e.g., a therapeutically-effective amount) incombination with a pharmaceutically acceptable carrier.

The terms “polymerase chain reaction” or “PCR” are art recognized andtypically refer to a reiterative amplification reaction catalyzed by athermostable polymerase for the purpose of increasing the number ofcopies of a target nucleic acid sequence (e.g., a DNA or RNA sequence)for detection or cloning.

The term “resistance of a cell to an anticancer agent” refers to the artrecognized phenomenon observed in cancer cells that have been exposed toa chemotherapeutic agent whereby the cell develops a tolerance orresistance to the growth inhibitory or killing properties of theanticancer agent.

The term “subject” is intended to include human and non-human animals(e.g., mice, rats, rabbits, cats, dogs, livestock, and primates).Preferred human animals include a human patient having a disordercharacterized by aberrant cell growth, e.g., a cancer.

The term “telomere” refers to the modified end of a eukaryoticchromosome which is frequently abnormally extended in a cancer cell.

The term “telomere damage-inducing” refers to any measurable change tothe end of a telomere when e.g., compared to a control cell, chromosome,or nucleic acid and includes chromosomal fragmentation, telomereshortening, and the presence of DNA free ends.

The term “telomerase” refers to the cellular enzyme or enzyme activitydirected to the nucleotide polymerization or maintenance of chromosomeends known as telomeres.

The term “telomerase inhibitory agent” refers to an agent that inhibits(completely or partially) the activity of the enzyme telomerase.

The term “therapeutically-effective amount” of a telomerase inhibitoryagent, telomere damage-inducing agent, and/or other chemotherapeuticrefers to the amount of such an agent which, alone or in combination, iseffective, upon single- or multiple-dose administration to the subject,e.g., a human patient, at inhibiting or reducing aberrant cell growth,e.g., a cancer.

The term “telomeric DNA” or “telomeric DNA fragment” refers to DNAderived from a cell comprising a telomere and existing as an intactpiece of DNA, e.g., as a chromosome, or, as a DNA fragment containing atelomere, which may be desired for experimental manipulation including,e.g., contacting with a probe or primer. The telomeric DNA may befragmented mechanically or enzymatically using, e.g., a nucleic, such asa restriction enzyme.

The term “timed-release formulation” refers to a formulation of a agent,wherein the agent is delivered to a site of interest in a form thateither becomes active, in a desirable period of time, e.g., in arelatively short period of time, e.g., a “quick release formulation”(e.g., within a few hours), or over a sustained period of time, e.g., a“slow release formulation” (e.g., from over 3 hours to several days).

Methods for Inhibiting or Reducing Cell Growth

In one aspect, the invention features methods for inhibiting or reducingcell growth, e.g., aberrant growth, e.g., hyperplastic or hypertrophiccell growth, by contacting the cells with at least one telomeredamage-inducing agent and at least one telomerase inhibiting agent. Ingeneral, the methods include a step of contacting pathologicalhyperproliferative cells (e.g., a cancer cell) with an amount of atleast one telomere-damage inducing agent and at least one telomeraseinhibiting agent which, in combination, is effective to reduce orinhibit the proliferation of the cell, or induce cell killing.

The present methods can be performed on cells in culture, e.g., in vitroor ex vivo, or can be performed on cells present in a subject, e.g., aspart of an in vivo therapeutic protocol. The therapeutic regimen can becarried out on a human or on other animal subjects. The enhancedtherapeutic effectiveness of the combination therapy of the presentinvention represents a promising alternative to conventional highlytoxic regimens of anticancer agents.

While either the cytotoxic or telomere damage-inducing agent or thetelomerase inhibitory agent can be utilized alone, the agents arepreferably combined for a synergistic effect. Even further, these agentsmay be further combined with other anticancer agents, e.g.,antimicrotubule agents, topoisomerase I inhibitors, topoisomerase IIinhibitors, antimetabolites, mitotic inhibitors, alkylating agents,intercalating agents, agents capable of interfering with a signaltransduction pathway (e.g., a protein kinase C inhibitors, e.g., anantihormone, e.g., an antibody against growth factor receptors), agentsthat promote apoptosis and/or necrosis, biological response modifiers(e.g. interferons, e.g. interleukins, e.g. tumor necrosis factors), orradiation.

Using the above strategy, the enhanced, and preferably synergisticaction of the telomere damage-inducing agent when used in combinationwith a telomerase inhibitory agent (and optionally, in furthercombination with another anticancer agent) improves the efficacy of theanticancer agent/s allowing for the administration of lower doses of oneor more of these anticancer agents (even, e.g., a subtherapeutic dose ofan agent if only tested or used alone rather than in combination) thusreducing the induction of side effects in a subject, such as a humancancer patient (e.g., any art recognized side effects associated withthe administration of an unmodified dose of a chemotherapeutic, e.g.,hair loss, neutropenia, thrombocytopenia, intestinal epithelial cellsloughing, etc.).

For example, in a preferred embodiment, the cytotoxic agent is presentin a lower dose, e.g., an amount equal to or lower than the one used inconventional chemotherapy. For example, for the combination ofpaclitaxel and carboplatin with an FGF antagonist, the dose ofpaclitaxel is equal to or below 225 mg/m², and the dose of carboplatinis chosen to achieve a total concentration-time product of equal to orbelow 6-7 mg/ml.min in previously untreated patients, or equal to orbelow 4-5 mg/ml.min in patients that have received chemotherapypreviously; the treatment is repeated every 3 weeks. For example, forthe combination of estramustine phosphate and taxotere with an FGFantagonist, the daily oral dose of estramustine is equal to or below1400 mg, and the dose of taxotere is equal to or below 70 mg/m² over 1hour; the treatment is repeated every 3 weeks. For example, for thecombination of doxorubicin and ketoconazole with an FGF antagonist, theweekly dose of doxorubicin is equal to or below 20 mg/m² by 24 hrinfusion, and the total daily oral dose of ketoconazole is equal to orbelow 1200 mg. For example, for the combination of cyclophosphamide,doxorubicin and 5-fluorouracil with an FGF antagonist, the dose ofintravenous cyclophosphamide is equal to or below 500 mg/m², the dose ofdoxorubicin is equal to or below 50 mg/m², and the dose of5-fluorouracil is equal to or below 500 mg/m²; the treatment is repeatedevery 4 weeks (the 5-fluorouracil dose is given once per week for twoweeks whereas the doses of doxorubicin and cyclophosphamide are givenonce every 4 weeks). For example, for the combination of Herceptin andcisplatin with an FGF antagonist, the Herceptin dose is equal to orbelow 250 mg on day 0, followed by 9 weekly doses of equal to or below100 mg, and the cisplatin dose is equal to or below 75 mg/m² on days 1,29, and 57. For example, for the combination of irinotecan with an FGFantagonist, the four weekly doses of irinotecan are equal to or below125 mg/m²; the treatment cycle is 4 weeks on and 2 weeks off. Forexample, for the combination of irinotecan with an FGF antagonist, thedose of irinotecan is 350 mg/m² every 3 weeks. For example, for thecombination of 5-fluorouracil and leucovorin with an FGF antagonist, thefive daily intravenous bolus doses of 5-fluorouracil are equal to orbelow 425 mg/m², and the five daily intravenous bolus doses ofleucovorin are equal to or below 20 mg/m²; the treatment cycle is 1 weekon and 4 weeks off. For example, for the combination of gemcitabine andcisplatin with an FGF antagonist, the three weekly doses of gemcitabineare equal to or below 1000 mg/m², and the single cisplatin dose given onday 2 is equal to or below 100 mg/m²; the treatment is repeated every 4weeks.

Methods for Treating Cancer

The methods of the invention can be used in treating malignancies of thevarious organ systems, such as those affecting lung, breast, lymphoid,gastrointestinal (e.g., colon), and the genitourinary tract (e.g.,prostate), pharynx, as well as adenocarcinomas which includemalignancies such as colon cancer, rectal cancer, renal cell carcinoma,prostate cancer and/or testicular tumors, non-small cell carcinoma ofthe lung, cancer of the small intestine, and cancer of the esophagus.

Exemplary solid tumors that can be treated include, e.g., fibrosarcoma,myosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, gastric cancer, esophageal cancer,colon carcinoma, rectal cancer, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, uterine cancer, cancer of the head andneck, skin cancer, brain cancer, squamous cell carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinoma,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicularcancer, lung carcinoma, small cell lung carcinoma, non-small cell lungcarcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma, retinoblastoma, leukemia, lymphoma, or Kaposi'ssarcoma.

The methods of the invention can also be used to inhibit or reduce thegrowth of a cell of hematopoietic origin, e.g., arising from themyeloid, lymphoid, or erythroid lineages, or any precursor cellsthereof. For instance, the present invention contemplates the treatmentof various myeloid disorders including, but not limited to, acutepromyeloid leukemia (APML), acute myelogenous leukemia (AML), andchronic myelogenous leukemia (CML) (reviewed in Vaickus, L. (1991) Crit.Rev. Oncol./Hemotol. 11:267-97). Lymphoid malignancies which can betreated by the method include, but are not limited, to acutelymphoblastic leukemia (ALL; which includes B-lineage ALL and T-lineageALL), chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL),hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM).

Additional forms of malignant lymphomas contemplated by the treatmentmethod of the present invention include, but are not limited to,non-Hodgkin's lymphoma and variants thereof, e.g., peripheral T-celllymphomas, adult T-cell leukemia/lymphoma (ATL), cutaneous T-celllymphoma (CTCL), and large granular lymphocytic leukemia (LGF).

Other malignancies which can be treated by the subject methods includeerythroleukemias, lymphomas, Hodgkin's disease, and malignancies ofuncertain origin, e.g., which are not easily categorized and may, e.g.,exhibit multiple cell types, such as certain embryonic carcinomas orteratomas.

For example, the subject can be a patient with non-small cell lungcancer, and is treated with a combination of paclitaxel, carboplatin andAZT, or with a combination of gemcitabine, cisplatin, and AZT.

In another example, the subject can be a patient with hormone refractoryprostate cancer, who is treated with a combination of estramustinephosphate, taxotere, and AZT, or with a combination of doxorubicin,ketoconazole, and AZT.

In still another example, the subject can be a patient with metastaticbreast cancer, who is treated with a combination of cyclophosphamide,doxorubicin, 5-fluorouracil, and AZT, or a combination of doxorubicin,taxotere, and AZT. In a related example, the subject is a patient withadvanced breast cancer that overexpresses the HER2/neu oncogene, who istreated with Herceptin and AZT, with or without paclitaxel or cisplatin.

In still another example, the subject can be a patient with advanced ormetastatic colorectal cancer, who is treated with a combination ofirinotecan and AZT. In a related example, the subject is a patient withadvanced colon cancer, who is treated with a combination of5-fluorouracil, leucovorin, and AZT.

Methods for Evaluating the Efficacy of an Agent

In another aspect, the invention features methods for evaluating theeffectiveness of an agent to serve as a telomere damage-inducing agentalone, or in combination with a telomerase inhibitor. The methodincludes contacting a cell with one or more agents and evaluating theability of the agent to induce telomere damage, e.g., telomereshortening, fragmentation, or the presence of chromosomal free ends,with the partial or overall telomere shortening being correlated witheffectiveness of treating a disorder, for example, a cancer.

Other related aspects encompassed by the invention include, combiningthe agents with known chemotherapeutic agents and using theassays/animal models disclosed herein, to determine if the agents arecompatible or contraindicated. Still further, the invention contemplatesusing such methods for determining the susceptibility of the cancer to agiven treatment and/or the resistance of the cancer to a giventreatment.

For example, methods disclosed herein can be used for determiningtelomere shortening, fragmentation, or the presence of chromosomal freeends when, for example, analyzing a sample, e.g., for the level of tumorsensitivity to the treatment with a combination of a telomeredamage-inducing agent/s and/or telomerase inhibitory agent/s. The methodincludes, evaluating the length of individual telomeres or the averagelength of telomeres in cells, and the effect of the telomeredamage-inducing agent on the telomere length. A short length of thetelomeres, or extensive shortening of the telomeres after treatment withthe telomere-damaging agent, would indicate sensitivity to the treatmentapproach.

Typically, this approach would be used on a naive cancer, i.e., a cancercell or tissue (e.g., a biopsy sample) that had never been exposed tothe test agents. Alternatively, the same methods may be employed fordetermining if a cancer that has acquired resistance to a particulartreatment protocol which may, e.g., have resulted from prolongedtreatment with a given chemotherapeutic regimen. Accordingly, the samemethods may be employed except that a non-naive cancer cell or tissuewould be used.

Still further, conducting both assays (i.e., a telomere damage-inducingassay and a telomerase inhibitory assay) for a given agent allows forthe identification of an agent which can have both desired activities.Moreover, conducting both assays allows for a determination of agentsynergy.

In a preferred embodiment, the cell sample is derived from a subject ortissue from a subject, e.g., a human patient having a disease ofaberrant cell, e.g., a cancer patient. The sample may be from, e.g., aprimary tumor, a metastatic tumor, a leukemia, or other cell sourcederived therefrom.

In a preferred embodiment the method includes choosing a particulartherapeutic combination, e.g. a particular anticancer treatment, e.g. aparticular cytotoxic agent, telomere damage-inducing agent, telomeraseinhibitory agent, or dosage or cocktail thereof, based on the length ofthe telomeres in the cell, sensitivity of the cells, and/or absence ofcell resistance to the therapy.

Methods for Prognosing/Diagnosing/Staging a Disorder

In another aspect, the invention also includes a method for treating asubject, preferably a human, that has been prognosed as likely, or aboutto have a cancer, or diagnosed as having a cancer. The method mayfurther involve staging the disorder. i.e., isolating a sample from thepatient having or about to have a cancer and using the assays of theinvention to determine if the cell is cancerous (e.g., expressing highamounts of telomerase), or likely to respond to the agents describeherein (or, e.g., determined to be resistant to the agents).

Methods for prognosing or diagnosing a subject for a cancer are wellknown in the art. In addition, genetic profiling may be performed todetermine if a given subject is at risk to developing a cancer bydetermining , e.g., if the subject has a mutation in a tumor suppressorgene (e.g., p53, Rb) or the presence of a cancer susceptibility gene(e.g., BRCA1, BRCA2).

In addition, methods of staging a disorder, e.g., a proliferativedisorder, e.g., a cancer in a subject, may be performed if desired. Themethod includes providing a sample, e.g., a cancerous sample, e.g., atissue, a bodily fluid, e.g., urine, blood, or CSF, or a biopsy, fromsaid subject, evaluating the expression of one or moretelomerase-related genes, e.g., by contacting said cancerous samplewith, a nucleic acid probe that selectively hybridizes to one or moretelomerase-related gene products whereby an increase in the level of oneor more telomerase-related gene products, relative to a control,indicates a stage in the disorder, e.g., that the cancer is benign,premalignant, or malignant (e.g., metastatic).

Telomere Damage-Inducing Agents and Methods for Their Identification

The invention provides telomere damage-inducing agents such as, e.g.,paclitaxel. Typically, telomere inducing agents are capable of causingdamage at the ends of eukaryotic chromosomes which can be measured as,e.g., a fragmentation of the chromosome, shortening of the ends of thechromosome, or by the detectable presence of an increase of freechromosomal ends.

Frequently, a telomere damage-inducing agent, such as paclitaxel, mayalso cytostatic or cytotoxic. Accordingly, various candidate telomeredamage-inducing agents may be, e.g., vincristine, vinblastine,vindesine, vinorelbin, taxotere (Docetaxel), topotecan, camptothecin,irinotecan hydrochloride (Camptosar), doxorubicin, etoposide,mitoxantrone, daunorubicin, idarubicin, teniposide, amsacrine,epirubicin, merbarone, piroxantrone hydrochloride, 5-fluorouracil,methotrexate, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate,cytarabine (Ara-C), trimetrexate, gemcitabine, acivicin, alanosine,pyrazofurin, N-phosphoracetyl-L-asparate (PALA), pentostatin,5-azacitidine, 5-Aza-2′-deoxycytidine, adenosine arabinoside (Ara-A),cladribine, ftorafur, UFT (combination of uracil and ftorafur),5-fluoro-2′-deoxyuridine, 5-fluorouridine, 5′-deoxy-5-fluorouridine,hydroxyurea, dihydrolenchlorambucil, tiazofurin, cisplatin, carboplatin,oxaliplatin, mitomycin C, BCNU (Carmustine), melphalan, thiotepa,busulfan, chlorambucil, plicamycin, dacarbazine, ifosfamide phosphate,cyclophosphamide, nitrogen mustard, uracil mustard, pipobroman,4-ipomeanol, dihydrolenperone, spiromustine, geldenamycin,cytochalasins, depsipeptide, Lupron, ketoconazole, tamoxifen, goserelin(Zoledax), flutamide,4′-cyano-3-(4-fluorophenylsulphonyl)-2-hydroxy-2-methyl-3′-(trifluoromethyl)propionanilide,Herceptin, anti-CD20 (Rituxan), interferon alpha, interferon beta,interferon gamma, interleukin 2, interleukin 4, interleukin 12, tumornecrosis factors, and radiation.

In one embodiment, the telomere damage-inducing agent is determinedusing, e.g., the methods of the invention, to be capable of causingtelomere damage.

In a preferred embodiment, the telomere damage-inducing agent is furthertested for telomerase inhibitory activity, or its ability to work inconcert with a another agent having telomerase inhibitory activity.

In a most preferred embodiment, the telomere damage-inducing agent isdetermined to work in concert with a telomerase inhibitory agent, morepreferably in synergy with the other agent such that the combination ofagents, when administered to a cancer cell (or an animal having acancer), is more effective at inhibiting or reducing the undesiredgrowth of the cancer cell then if either one of the agents wasadministered alone. In a preferred embodiment, the combinedeffectiveness of a telomere damage-inducing agent and a telomeraseinhibitory agent is more than additive, e.g., synergistic. As disclosedherein, the invention provides methods for determining such synergy.

The telomere damage-inducing agents used in the methods and compositionsof the present invention can be, e.g., a small molecule, macromolecule(e.g., peptide, polypeptide, or antibody), or even, e.g., a nucleic acid(e.g., plasmid vector, recombinant virus, antisense nucleic acid ).Accordingly, the invention encompasses agents such as a “gene therapyconstruct” useful for gene therapy purposes, in treatments forinhibiting or reducing the growth of a cell, preferably aberrant growth,e.g., a cancer, that may be either genetic or acquired.

The general approach of gene therapy involves the introduction of anucleic acid into cells such that one or more gene products encoded bythe introduced genetic material is produced in the cells to produce agene product that can result in an increase in the desired activity,e.g., telomere damage or telomerase inhibition. Such a gene product canbe, e.g., a nuclease, such as a restriction endonuclease. Alternatively,the gene product may function any where in a pathway that leads to thedesired activity, e.g., telomere damage, or telomerase inhibition. Forreviews on gene therapy approaches see Anderson, W. F. (1992) Science256:808-813; Miller, A. D. (1992) Nature 357:455-460; Friedmann, T.(1989) Science 244:1275-1281; and Cournoyer, D., et al. (1990) Curr.Opin. Biotech. 1:196-208.

Gene therapy applications of particular interest in cancer treatmentinclude the overexpression of a telomere damage-inducing agent ortelomerase inhibitor (or both) and such an agent can be either antisensebased or encode a gene product, e.g., a polypeptide. Preferably, thegene of interest is selectively or inducibly expressed, e.g., at aparticular time (e.g., upon the administration of an inducing agent)and/or in a particular cell type or tissue. Examples of inducible ortissue specific expression vectors are well known in the art (see alsoExample 7).

Telomerase Inhibitory Agents and Methods for Their Identification

The invention provides telomerase inhibitory agents such as, e.g., thenucleotide analog, AZT or, e.g., an inhibitory nucleic acid such as anantisense nucleic acid. Typically, telomerase inhibitory agents such as,e.g., AZT, also disrupt DNA replication. Accordingly, various nucleotideanalogs having such activity, as well as known telomerase inhibitors,are encompassed by the invention, and they include those agents recitedin, e.g., Table 1. TABLE 1 Telomerase Inhibitory Agents Category NameSelected References Reverse AZT Beltz et al., (1999) AnticancerTranscriptase Res. 19: 3205-3211; Yegorov, inhibitor et al., (1999)Anticancer Drug Des., 14: 305-3016; Multani, et al. (1998) Clin. Cancer.Res. 4: 2569-2570; Gomez et al. 91998) Biochem Biophys Res Commun 246:107-110; Kondo et al. (1998) Oncogene16: 2243-2248 d4T Beltz et al.,(1999) Anticancer Res. 19: 3205-3211 carbovir Yegorov, et al., (1999)Anticancer Drug Des., 14: 305- 3016 7-deaza-dGTP Fletcher et al. (1999)Biochem Biophys Res Commun 265: 51- 56, Fletcher et al. (1996)Biochemistry 35: 15611-15617 7-deaza-dATP Fletcher et al. (1996)Biochemistry 35: 15611-15617 Antisense PNA Shammas et al. (1999)Oncogene 18: 6191-6200; Herbert et al. (1999) Proc Natl Acad Sci USA 96:14276-14281 2′-O-MeRNA Herbert et al. (1999) Proc Natl Acad Sci USA 96:14276-14281 phosphodiester oligo phosphorothioate Oligo DifferentiationVitamine D3 Yamada et al. (1998) Leuk Res Agents 22: 711-7 retinoic acidYamada et al. (1998) Leuk Res 22: 711-7 hemin Yamada et al. (1998) LeukRes 22: 711-7 Tamoxifen PKC inhibitors: Ku et al. (1997) Biochembisindolyl- Biophys Res Commun 241: maleimide 730-736 I and H-7 Othersporphyrins TMPyP4 Izbivka et al., (1999) Anticancer Drug Des. 14: 355-365; Izbicka et al. (1999) Cancer Res 59: 639-644 disubstituted Harrisonet al. (1999) Bioorg acridine Med Chem Lett 9: 2463: 2468 CyclooxygenaseJTE-522 Nishimura et al. (1999) Jpn J inhibitor Cancer Res 90: 1152-1162rhodacyanine FJ5002 Naasani et al. (1999) Cancer Res 59: 4004-4011fluorenone Perry et al. (1999) J Med Chem derivatives 42: 2679-2684 teacatechins Naasani (1998) Biochem Biophys Res Commun 249: 391-196 TPAantisense See text.

In one embodiment, the telomerase inhibitory agent is determined using,e.g., the methods of the invention, to be capable of inhibiting telomererepair. Accordingly, a candidate compound, e.g., a cytostatic orcytotoxic compound, can be tested for telomerase inhibitory activity.

In a preferred embodiment, the telomerase inhibitory agent is furthertested for telomere damage-inducing activity or its ability to work inconcert with a separate agent having telomere damage-inducing activity.

In a most preferred embodiment, the telomerase inhibitory agent isdetermined to work in concert with a telomere damage-inducing agent,more preferably in synergy with the other agent such that thecombination of agents, when administered to a cancer cell (or an animalhaving a cancer), is more effective at inhibiting or reducing theundesired growth of the cancer cell then if either one of the agents wasadministered alone. In a preferred embodiment, the combinedeffectiveness of a telomerase inhibitory agent and a telomeredamage-inducing agent is more than additive, e.g., synergistic. Asdisclosed herein, the invention provides methods for determining suchsynergy.

The telomerase inhibitory agents used in the methods and compositions ofthe present invention can be, e.g., any of the molecule types describedabove for telomere damage-inducing agents and therefore include, e.g., asmall molecule, macromolecule (e.g., peptide, polypeptide, or antibody),nucleic acid (e.g., plasmid vector, recombinant virus, antisense) andgene therapy approaches (see, e.g., Example 6).

A determination of telomerase inhibitory activity using any of themethods described in, e.g., U.S. Pat. Nos. 5,645,986; 5,972,605; and6,007,989 may also be performed or combined with the foregoing methods.

Other telomerase inhibitors include, e.g., the reverse transcriptaseinhibitors Abacavir (1592U89), Adefovir dipivoxil, ADAM, AF/ABDP,(Alkylamino) piperidine bis heteroaryl)piperizine, Alterperylenol,Atevirdine mesylate (Bisheteroypiperazine, U-87201-E, AZT (Zidovudine),GAZT, AZTMP, AZTTP, 3-benzene-sulphonyl-5-chloroindol-3-carboxamide(L-737, 126), BHAP, 2-chloro-5-(2-methyl-5,6-dihydro-1,4-oxathin-3-ylcarboxamido) isopropylbenzoate (NSC 615985, UC84),5-Chloro-2′,3′-dideoxy-3′-fluorouridine (935U83),7-deaza-2′-deoxypurines, ddA (Didanosine), ddl, F-ddl, ddG, ddC(Zalcitabine), DDCN, Bz-DDCN, Delavirdine, Delavirdine mesylate(U-90152T), 1-(2′,6′-difluorophenyl)-1H,3H-thiazolo[3,4-a]benzimidazol,5,8-dimethoxy-3-hydroxy-4-quinolone, 2′-deoxy-3′-oxa-4′-thiocytidine(BCH-10652), 1,5-diphenylpyrazole, Dipyridodiazepinones, BI-RG-587,1-(2,6-difluorophenyl)-1H, 3H-thiazolo[3,4-a]benzimidazol (TBZ NSC625487), Thiazolobenzimidazol (NSC 625487),3-(5-Dimethylamino-1-naphthalenesulphonyl)-2-(3-pyridyl)thiazolidine(YHI-1), Efavirenz (SUSTIVA, DMP266, EFV),2′-fluoro-2′,3′-dideoxyarabinosyladenine (F-ddA), HBY097,1-[2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT), Imidazol,L-696,229, L-697,661, L-697,639, L-734,005, L-738,372, Lamivudine,MKC-442, Nevirapine, PNU-142721, Phenylethylthiazolylthiourea (PETT),(s4dU)35, Suramin, TDA, RD4-2040, Thiocarboxanilide, UC-781, U-88204E,UC38, UC84, UC040, UC82, UC781, uridine-3′-spiroxirane.

In one embodiment, other telomerase inhibitory agents include antisensemolecules such as, e.g. directed against the template RNA of telomerase,or telomere mimic oligomers, or ribozymes, with different backbones,PNA, 2′-O-MeRNA, phosphodiester oligonucleotide, phosphorothioateoligonucleotide.

In another embodiment, other telomerase inhibitors includedifferentiation agents such as, e.g., vitamin D3, retinoic acid, andhemin.

In another embodiment, telomerase inhibitory agents include, e.g., PKCinhibitors that indirectly inhibit telomerase action, e.g.,bisindolylmaleimide I, 9-hydroxyelipticine, H-7.

In still another embodiment, telomerase inhibitory agents include ,e.g., porphyrins, TMPyP4, acridine analogs, JTE-522, rhodacyanine,FJ5002, fluorenone derivatives, tea catechins, TPA, epigallocatechingallate (EGCG), Rhodacyanines (FJ5002), the fungal metabolitealterperynol, 3,3′-diethyloxadicarbocyanine, and patented inhibitors(U.S. Pat. No. 5,863,936) Nihon Kayaku: Japanese Patent # 11-60573)

Screening Methods

The present invention provides a method of identifying an agent capableof inhibiting or reducing the growth of a cell by contacting a cell witha candidate agent and determining if the agent has any telomeredamage-inducing activity and/or telomerase inhibitory activity. Theassays disclosed herein allow for the selection of an agent or agents,when used alone or in combination, that are capable of inducing telomeredamage and reducing telomerase activity for the purpose of inhibiting orreducing the growth of a cell, e.g., a cancerous cell.

It will be appreciated by the skilled artisan that, given the teachingsdisclosed herein, it will be only a matter of routine experimentation totest a known agent, or screen for a candidate agent, having either ofthe above-mentioned activities. An advantage of the invention is thatthe testing/screening assays of the present invention are also amenableto a high throughput format for the efficient analysis of, e.g., dosagesand formulations and/or the screening of multiple agents in the form of,e.g., a library of molecules. In particular, these assays allow for thescreening and identification of agents that are capable of functioningin synergy. Accordingly, these assays allow for an efficientdetermination if one or more of the agents may be delivered at asubtherapeutic dose because of the agents' resultant synergy when usedin combination. This is especially advantageous for being able to reducedosage in order to reduce or eliminate any undesired side effects thatmay be caused by the agents when used alone or at conventional dosagelevels.

It is understood that the agents (including agents contained in acombinatorial library) may be small molecules, macromolecules (peptides,polypeptides), or even nucleic acids that exhibit a desired activity(i.e., telomere damage-inducing activity or telomerase inhibitoryactivity) by, e.g., encoding a gene product or by engaging in aninhibitory antisense hybridization reaction.

The invention is also designed to encompasses an agent or agentsidentified according to the foregoing screening methods for use in theinhibition or reduction in the growth of a cell, e.g., for the treatmentof a cancer. Still further, the agents so identified may be packagedinto various formulations for timed-release, tissue tropism, addedtherapeutic value, or ease of administration and these properties arediscussed in the following subsections.

Preferably, therefore, the assay according to the invention iscalibrated in the absence of the agent or agents to be tested, or in thepresence of a reference compound of known activity (e.g., telomeredamage-inducing activity and/or telomerase inhibitory activity) as areference value. For example, the agents disclosed herein, i.e.,paclitaxel which has telomere damage-inducing activity, and AZT, or anantisense corresponding to a telomerase, either of which has telomeraseinhibitory activity, can be used, either alone or in combinationdepending on whether the measure of one activity or a measure of agentsynergy, is desired.

A candidate telomere damage-inducing agent and/or telomerase inhibitoryagent of the present invention can be identified among any of the agentsdescribed herein or from a library of agents either known or unknownusing the assays described herein, preferably a cell based assay isused, e.g., as described in the examples, in combination with any of thenumerous approaches involving combinatorial library methods known in theart. For example, methods for the synthesis of molecular libraries canbe found in the art, for example in: DeWitt et al. (1993) Proc. Natl.Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and in Gallop et al. (1994) J. Med. Chem. 37:1233.

Thus, a library of agents, e.g., agents that are protein based,carbohydrate based, lipid based, nucleic acid based, natural organicbased, synthetically derived organic based, or antibody based compounds,can be assembled and assayed. In addition, high throughput assays may bedesirable in order to maximize the number of agents surveyed in a givenperiod of time.

Accordingly, it is within the scope of this invention to further use anagent, e.g., a telomere damaging-inducing agent or telomerase inhibitoryagent identified as described herein in an appropriate animal model. Forexample, an agent identified as described herein can be used in ananimal model to determine the efficacy (e.g., synergy), toxicity, orside effects of treatment with such an agent/s. Alternatively, an agentidentified as described herein can be used in an animal model todetermine the mechanism of action of such an agent. In addition, such anagent if deemed appropriate, may be administered to a human subject,preferably a subject having, or at risk for, a cancer.

The present invention also pertains to uses of novel agents identifiedby the above-described screening assays for diagnoses, prognoses, andtreatments of any of the disorders described herein. Accordingly, it iswithin the scope of the present invention to use such agents in thedesign, formulation, synthesis, manufacture, and/or production of a drugor pharmaceutical composition for use in the diagnosis, prognosis, ortreatment of any of the disorders described herein.

Methods of Administration

In one embodiment of the invention, the telomere damage-inducing agentand the telomerase inhibitory agent, are administered at the same timeor in overlapping time periods. Alternatively, the agents can beadministered at different times, where, e.g., one agent is administeredfirst while the other agent administered subsequently, or in reverseorder if desired.

In a preferred embodiment, the telomere damage-inducing agent and/or thetelomerase inhibitory agent is administered systemically. For example,the selected agent can be administered parenterally (e.g.,subcutaneously, intravenously, intramuscularly, intraperitoneally,intradermally, intravesically (i.e., urinary bladder), intrathecally,etc.), orally, nasally, rectally, topically, and/or transdermally.

In another embodiment, the telomere damage-inducing agent and/ortelomerase inhibitory agent is administered locally or regionally, usingany of the foregoing routes of administration.

In another embodiment, the method further includes repeated dosages ofthe same, or a different agent, and such particulars are furtherdiscussed below.

Dosage Regimens

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the agent or agents(e.g., in the form of a pharmaceutical composition) required. Forexample, the physician or veterinarian may typically start doses of theagents of the invention at levels lower than that required in order toachieve the desired therapeutic effect and gradually increase the dosageuntil the desired effect is achieved.

In general, a suitable dose of an agent of the invention will be thatamount of the agent which is the lowest dose effective to produce atherapeutic effect; i.e., treat a condition in a subject, e.g., cancer.Such an effective dose will generally depend upon the factors describedabove. Generally, intravenous and subcutaneous doses of the agents ofthis invention for a patient, will range from about 0.0001 to about 100mg per kilogram of body weight, more preferably from about 0.01 to about10 mg per kg, and still more preferably from about 0.10 to about 4 mgper kg. If desired, the effective daily dose of the active agent may beadministered as two, three, four, five, six, or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms.

While it is possible for an agent of the present invention to beadministered alone or in combination with another agent, it ispreferable to administer the agent/s as a pharmaceutical composition.

In a preferred embodiment, the agent is present in an amount lower thanthe one used in conventional chemotherapy. For example, for thecombination of paclitaxel and carboplatin with a telomerase inhibitoryagent, the dose of paclitaxel is below 135 mg/m2, and the dose ofcarboplatin is chosen to achieve a total concentration-time product ofbelow 6-7 mg/ml.min in previously untreated patients, or below 4-5mg/ml.min in patients that have received chemotherapy previously.

For example, for the combination of estramustine phosphate and taxoterewith a telomerase inhibitory agent, the daily oral dose of estramustineis below 1400 mg, and the dose of taxotere is below 70 mg/m2 over 1hour.

For example, for the combination of doxorubicin and ketoconazole with atelomerase inhibitory agent, the dose of doxorubicin is below 20 mg/m2by 24 hr infusion, and the total daily oral dose of ketoconazole isbelow 1200 mg.

For example, for the combination of cyclophosphamide, doxorubicin, and5-fluorouracil with a telomerase inhibitory agent, the dose ofintravenous cyclophosphamide is below 500 mg/m2, the dose of doxorubicinis below 50 mg/m2, and the dose of 5-fluorouracil is below 500 mg/m2.

For example, for the combination of herceptin and cisplatin with atelomerase inhibitory agent, the herceptin dose is below 250 mg on day0, followed by 9 weekly doses of below 100 mg, and the cisplatin dose isbelow 75 mg/m2 on days 1, 29, and 57.

For example, for the combination of irinotecan with a telomeraseinhibitory agent, the four weekly doses of irinotecan are below 100-125mg/m2.

For example, for the combination of 5-fluorouracil and leucovorin with atelomerase inhibitory agent, the five daily intravenous bolus doses of5-fluorouracil are below 425 mg/m2, and the five daily intravenous bolusdoses of leucovorin are below 20 mg/m2.

For example, for the combination of gemcitabine and cisplatin with atelomerase inhibitory agent, the three weekly doses of gemcitabine arebelow 1000 mg/m2, and the single cisplatin dose given on day 2 is below100 mg/m2.

Compositions and Formulations

In another aspect, the invention features, a pharmaceutical compositionwhich includes, at least one agent that causes telomere damage, at leastone telomerase inhibitory agent, and a pharmaceutically acceptablecarrier. Alternatively, the agents may be formulated separately.Preferably, the agent/s are present in an amount effective to enhancethe efficacy of, e.g., an additional cytotoxic agent, in reducing orinhibiting the proliferation, or in enhancing the killing, of ahyperproliferative cell.

In a preferred embodiment, the pharmaceutical composition orcompositions are packaged with instructions for use as described herein.

In a preferred embodiment, the agent that induces telomere damage ischosen from those disclosed herein, e.g. paclitaxel, or derivativethereof.

In a preferred embodiment, the telomerase inhibitory agent is chosenfrom those disclosed herein, e.g., AZT, or an antisense nucleic acidcorresponding to telomerase (see, e.g., Table 1).

The invention also encompasses timed-release formulations. For example,a quick release formulation of a telomere damage-inducing agent and/ortelomerase inhibitory agent or a slow release formulation of a telomeredamage-inducing agent and/or telomerase inhibitory agent, and apharmaceutically acceptable carrier.

In one embodiment, the telomere damage-inducing agent is paclitaxel. Forexample, one quick release formulation advantageously releases about 50nM of paclitaxel over about 1 hour or less.

In another embodiment, the pharmaceutical composition is suitable forintravenous injection. The composition may also be suitable for local,regional, or systemic administration.

In another embodiment, the pharmaceutical composition may comprise oneor more pharmaceutically acceptable carriers. In yet another embodiment,the invention pertains to nanoparticles, which comprise a cross linkedgelatin and a therapeutic agent, e.g., telomere damage-inducing agentand/or telomerase inhibitory agent, such as, for example, paclitaxel,and/or AZT. In a further embodiment, the invention pertains to acompositions containing the nanoparticles and a pharmaceuticallyacceptable carrier. The carrier can be, for example, suitable forsystemic, regional, or local administration. In another embodiment, theinvention pertains to a method of treating a patient comprisingadministering the nanoparticles of the invention. In one embodiment, thenanoparticles are about 500 nm to about 1 μm, or about 600 nm to about800 nm in diameter.

The invention also pertains to microparticles comprising a therapeuticagent, e.g., telomere damage-inducing agent and/or telomerase inhibitoryagent, such as paclitaxel and/or AZT. In one embodiment, themicroparticle is about 500 nm to about 100 μm, about 500 nm to about 50μm, about 500 nm to about 25 μm, about 500 nm to about 20 μm, about 500nm to about 15 μm, about 500 nm to about 10 μm, about 750 nm to about 10μm, about 1 μm to about 10 μm, about 750 nm to about 7.5 μm, about 1 μmto about 7.5 μm, about 2 μm to about 7.5 μm, 3 μm to about 7 μm, orabout 5 μm in diameter. In another embodiment, the invention pertains toa composition which comprises the microparticles and a pharmaceuticallyacceptable carrier. The pharmaceutically acceptable carrier may be, forexample, suitable for administration to a patient locally, regionally,or systemically. The invention also pertains to a method for treating apatient, comprising administering to the patient microparticles of theinvention and a pharmaceutically acceptable carrier.

In another embodiment, the invention features a microparticle suitablefor administration to a patient locally, regionally, or systemically,comprising paclitaxel, wherein said microparticle has a diameter ofabout 5 μm. In another further embodiment, the invention featuresmicroparticles suitable for administration to a patient locally,regionally, or systemically, comprising AZT, wherein said microparticlehas a diameter of about 5 μm.

The invention also pertains to a kit, i.e., an article of manufacture,for the treatment of a cancer. The kit contains an telomeredamage-inducing agent and/or telomerase inhibitory agent in apharmaceutically acceptable carrier, a container, and directions forusing said telomere damage-inducing agent and/or telomerase inhibitoryagent for inhibiting or reducing the growth of a cell, e.g., aberrantgrowth associated with, e.g., a cancer or a tumor. For example, a kit ofthe invention may comprise a telomere damage-inducing agent and atelomerase inhibitory agent for previous, subsequent, or concurrentadministration. The kit may also provide the telomere damage-inducingagent and/or telomerase inhibitory agent formulated in dosages andcarriers appropriately for local, regional, or systemic administration.Still further, the kit may also provide for the prognosing, diagnosing,and/or staging of a cancer for, e.g., determining the susceptibility orresistance of the cancer.

Pharmaceutical compositions comprising compounds of the invention maycontain wetting agents, emulsifiers and lubricants, such as sodiumlauryl sulfate and magnesium stearate, as well as coloring agents,release agents, coating agents, sweetening, flavoring, and perfumingagents, and preservatives.

Formulations of the present invention include those suitable for oral,nasal, topical, transdermal, buccal, sublingual, rectal, vaginal, and/orparenteral administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. The amount of active ingredient which canbe combined with a carrier material to produce a single dosage form willgenerally be that amount of the agent which produces a therapeuticeffect. Generally, out of one hundred per cent, this amount will rangefrom about 1 per cent to about ninety-nine percent of active ingredient,preferably from about 5 per cent to about 70 per cent, most preferablyfrom about 10 per cent to about 30 per cent.

Methods of preparing these formulations or compositions include the stepof bringing into association an agent of the present invention with thecarrier and, optionally, one or more accessory ingredients. In general,the formulations are prepared by uniformly and intimately bringing intoassociation a compound of the present invention with liquid carriers, orfinely divided solid carriers, or both, and then, if necessary, shapingthe product.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of thepresent invention as an active ingredient. An agent of the presentinvention may also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules, and the like),the active ingredient is mixed with one or more pharmaceuticallyacceptable carriers, such as sodium citrate or dicalcium phosphate,and/or any of the following: fillers or extenders, such as starches,lactose, sucrose, glucose, mannitol, and/or silicic acid; binders, suchas, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; humectants, such as glycerol;disintegrating agents, such as agar-agar, calcium carbonate, potato ortapioca starch, alginic acid, certain silicates, and sodium carbonate;solution retarding agents, such as paraffin; absorption accelerators,such as quaternary ammonium compounds; wetting agents, such as, forexample, cetyl alcohol and glycerol monostearate; absorbents, such askaolin and bentonite clay; lubricants, such a talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof; and coloring agents. In the case of capsules,tablets and pills, the pharmaceutical compositions may also comprisebuffering agents. Solid compositions of a similar type may also beemployed as fillers in soft and hard-filled gelatin capsules using suchexcipients as lactose or milk sugars, as well as high molecular weightpolyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of the agents of theinvention include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups, and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluent commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Formulations of the pharmaceutical compositions of the invention forrectal or vaginal administration may be presented as a suppository,which may be prepared by mixing one or more compounds of the inventionwith one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active compound.

Formulations of the present invention which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate.

Dosage forms for the topical or transdermal administration of a compoundof this invention include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The active compound maybe mixed under sterile conditions with a pharmaceutically acceptablecarrier, and with any preservatives, buffers, or propellants which maybe required.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients, such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the present invention to the body. Such dosageforms can be made by dissolving or dispersing the compound in the propermedium. Absorption enhancers can also be used to increase the flux ofthe compound across the skin. The rate of such flux can be controlled byeither providing a rate controlling membrane or dispersing the activecompound in a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this invention.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more compounds of the invention incombination with one or more pharmaceutically acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containbuffers, bacteriostats, solutes which render the formulation isotonicwith the blood of the intended recipient or suspending or thickeningagents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption such as aluminum monostearate andgelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

The preparations of the present invention may be given orally,parenterally, topically, rectally, intralesionally, intraorbitally,intracapsularly, directly instilled into a cavity, or by inhalation.They are of course given by forms suitable for each administrationroute. For example, they are administered in tablets or capsule form, byinjection, inhalation, eye lotion, ointment, suppository, etc.administration by injection, infusion or inhalation; topical by lotionor ointment; and rectal by suppositories.

These compounds may be administered to humans and other animals fortherapy by any suitable route of administration, including orally,nasally, as by, for example, a spray, rectally, intravaginally,parenterally, intracisternally and topically, as by powders, ointmentsor drops, including buccally and sublingually.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms by conventional methodsknown to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compound employed, the age, sex, weight, condition, generalhealth and prior medical history of the patient being treated, and likefactors well known in the medical arts.

EXEMPLIFICATION OF THE INVENTION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

Throughout the examples, unless otherwise indicated, the practice of thepresent invention will employ conventional techniques of chemistry,molecular biology, microbiology, recombinant DNA technology, cellculture, and animal husbandry, which are within the skill of the art andare explained fully in the literature. See, e.g., Sambrook, Fritsch andManiatis, Molecular Cloning: Cold Spring Harbor Laboratory Press (1989);DNA Cloning, Vols. 1 and 2, (D. N. Glover, Ed. 1985); Harlow and Lane,Antibodies: a Laboratory Manual, (1988) Cold Spring Harbor;Oligonucleotide Synthesis (M. J. Gait, Ed. 1984); Nucleic AcidHybridization (B. D. Hames and S. J. Higgins, Eds. 1984); the seriesMethods In Enzymology (Academic Press, Inc.), particularly Vol. 154 andVol. 155 (Wu and Grossman, Eds.; and Current Protocols in MolecularBiology, eds. Ausubel et al. , John Wiley & Sons (1992)).

Materials and Methods:

The reagents and experimental protocols used in the appended examplesare briefly described below.

Chemicals and reagents. Drugs were obtained from the National CancerInstitute (Bethesda, Md.) or commercial sources. Cell Death DetectionELISA kit, and digoxigenin-labeled human telomeric probe from Oncor.Cefotaxime sodium was purchased from Hoechst-Roussel PharmaceuticalsInc. (Somerville, N.J.), and all other culture supplies from Gibco BRL(Grand Island, N.Y.). ApoAlert CPP32 assay kit and ApoAlert Annexin VApoptosis kit were purchased from Clontech Laboratories Inc (Palo Alto,Calif., C2.10 PARP monoclonal antibody from Enzyme Systems (Livermore,Calif, cell death detection ELISA kit from Boehringer Mannheim(Indianapolis, Ind.), chemiluminescent Western blot kit from Amersham(Arlington Heights, Ill.), and horseradish peroxidase-conjugated goatanti-mouse immunoglobulin from Dako Corp (Carpinteria, Calif. Allchemicals and reagents were used as received.

Tumors and cultures. Human cancer cell lines including breast MCF7cells, pharynx FaDu cells, prostate PC3 cells, ovarian SKOV3 cells,bladder RT4 cells, and lung A549 cells were purchased from American TypeCulture Collection (Manassas, Va.). The mdrl-transfected subclones ofMCF7 cells, i.e., BC19 cells, were obtained from Dr. Kenneth Cowan(National Cancer Institute, Bethesda, Md.). FaDu cells were maintainedin MEM, PC3 cells in RPMI-1640, SKOV3 in McCoy medium, MCF7 in eitherRPMI-1640 or MEM, and BC19 cells in RPMI 1640 medium. All culture mediawere supplemented with 9% heat-inactivated FBS and 0.1% 10 mMnon-essential amino acids, 2 mM L-glutamine, 90 μg/ml gentamicin and 90μg/ml cefotaxamine sodium. Cells were incubated with complete medium at37° C. in a humidified atmosphere of 5% CO₂ in air. For experimentscells were harvested from preconfluent cultures and resuspended in freshmedium. Cells were seeded in a 96-well microtiter plate and allowed toattach overnight (20-24 hr).

Establishment of human xenograft tumors in mice. The human pharynx FaDutumor xenograft was established as follows. FaDu cells with greater than90% viability, as determined by trypan blue exclusion, were used fortumor implantation. Cells were centrifuged and resuspended in Matrigel(1:1 v/v). Matrigel is a solubilized tissue basement membranepreparation extracted from the Engelbreth-Holmswarm mouse tumor and hasbeen shown to support the growth of human tumors in immunodeficient mice(Kleinman H et al. (1990) Proc Am Assoc Cancer Res 31:490-491). Thetumor establishment was achieved by subcutaneously injecting 10⁶ cells(0.1-0.2 ml) with an 18 gauge needle at left and right sides of theupper back. The tumor was removed when it reached a size of 0.5 to 1 gand used for experiments.

Histocultures of solid tumors. Histocultures are cultures of tumorfragments where the three dimensional structure and the heterogeneoustissue composition are maintained. Histocultures of the FaDu xenografttumors were established as described (Kuh, et al. (1999) J PharmcolExper Therap 290:871-880). Tumor specimens were washed three times withculture medium for three times and dissected into about 1 mm³ fragmentsunder sterile conditions. The culture medium for the FaDu histocultureswas MEM supplemented with 9% heat-inactivated FBS, 2 mM glutamine, 90μg/ml gentamicin and 90 μg/ml cefotaxime sodium.

Drug solutions. Stock solutions of paclitaxel were prepared in 70%ethanol at a concentration of 1 mM and stored at −70° C. Aliquots of thestock solution were added to the media such that the final concentrationof ethanol was <0.1% which experimentally had no effect on the cellproliferation. Stock solutions of 3′-azido-deoxythymidine (AZT) and3′-deoxy-2′,3′-didehydrothymidine (d4T) were prepared in doubledistilled sterile water at a concentration of 10 mM.

In vitro drug activity evaluation. Drug effect was measured in threeways, i.e., by enumerating the number of cells that remained attached inculture flask, by enumerating the number of cells that detached from thegrowth surface, and changes in levels of apoptosis. The firstmeasurement represents the overall drug effect, i.e., the combination ofcytostatic and cytotoxic effects, whereas the latter two measurementsreflect the cytotoxic effect. The remaining cell number after drugtreatment was measured using the sulforhodamine B (SRB) assay whichstains for cellular protein (Skehan P et al. (1990) J Natl Cancer Inst82:1107-1112).

In brief, cells were seeded onto 96-well plates. To avoid changes indrug concentration caused by excessive evaporation of medium in wellslocated at the edge of the plate only the inner wells in 96-well plateswere used. The outer wells contained only tissue culture media to serveas blanks. At the end of drug treatment, the culture medium was removedfrom the wells by aspiration and the cells were then fixed by incubatingwith 0.2 ml 10% trichloroacetic acid at 4° C. for 1 hr, followed by fivewashes with distilled water. The plates were then air-dried after whichSRB solution (0.05 ml of 0.4%) was added to stain cells for 10 min atroom temperature. Afterward, each plate was rinsed five times with 1%glacial acetic acid and allowed to air-dry. Tris-HCI (0.2 ml, 10 mM) wasthen added to each well to dissolve the SRB bound to cellular protein.The amount of SRB was measured by absorbance at 490 nm using an EL 340microplate biokinetics reader (Bio-Tek Instruments, Inc., Winooski,Vt.). The absorbance is proportional to the number of cells attached tothe culture plate. Each experiment used six replicates.

To determine the extent of cell detachment after drug treatment, 5.0×10⁵cells were seeded into 75 cm² culture flasks (Becton Dickinson, FranklinLakes, N.J.) and allowed to attach overnight (20-24 hr) in drug-freemedia prior to the initiation of drug treatment. Upon drug treatment,the supernatant along with two subsequent Versene rinses, whichcontained the detached cells, were collected. The cells that remainedattached to the growth surface were harvested by trypsinization. Thesesamples were centrifuged at 1500 rpm for 10 min and the resulting cellpellets were resuspended in 2 ml of culture media. The cell number inthe detached and attached fractions were counted using a Coulter counter(Coulter Electronic, Inc., Hialeah, Fla.).

Apoptosis was measured as the release of DNA-histone complex from thenucleus to the cytoplasm, using the Cell Death Detection ELISA kit whichquantifies the amount of DNA-histone complex released into thecytoplasm. Briefly, drug-treated cells were collected and lysed in thelysis buffer. The cytoplasmic fractions of the lysates were placed inwells of a 96-well microtiter plate pre-coated with mouse antihistoneprimary antibody and mouse anti-DNA-antibody conjugated to peroxidase.The peroxidase substrate, 2,2′-azido-di-[3-ethylbenzthiazolinesulfonate], was applied and the absorbance at 405 nm was measured.

Fluorescent in situ hybridization (FISH). Fluorescent in situtybridization (FISH) was used to identify the presence and to estimatethe approximate length of individual telomere structures at the end ofchromosomes. The FISH method used was a modification of a previouslypublished method (Multani AS et al. (1996) Anticancer Res 16: 3435-3438;Henderson S et al. (1996) J Cell Biol 134:1-12), and consists of thefollowing three steps: (a) the cells are fixed in a manner that promotesspreading of the chromosomes, (b) a nucleotide sequence complementary tothe telomere sequence is used as probe to bind to the telomeres and toprovide a point of attachment for fluorescently-labeled antibodies, and(c) the fluorescent signal is identified using fluorescent microscopy.

The methods are as follows. To spread the chromosomes, the cell pelletwas incubated with a hypotonic solution (75 mM KCl) at 37° C. for 20min, washed three times and fixed with acetic acid and methanol, droppedonto slides, air-dried, and denatured in 2×SSC and 70% formamide at 74°C. for 5 min. To detect the telomeres, the cells were incubated with adigoxigenin-labeled human telomeric probe (Oncor) at 37° C. for 16 to 18hr. The slides were washed consecutively in 2×SSC at 42° C. and 72° C.,each time for 5 min. After blocking with 5% skim milk for 10 min, cellswere incubated sequentially with rhodamine-labeled (red color)anti-digoxigenin, rabbit anti-sheep, and rhodamine-labeled anti-rabbitantibodies, at room temperature for 30 min. Between each step, theslides were washed three times in 130 mM phosphate buffer, pH 7.4, 0.1%Triton X-100, each time for 5 min. The chromosomes were thencounterstained with 0.1 μg/ml of 2,4-diamidine-2-phenylindole (bluecolor) and examined under a fluorescence microscope at 100×magnification using a triple band pass filter at 340 nm.

Caspase Activity. The activity of Caspase 3 was measured using theApoAlert CPP32 assay (Clontech Laboratories, Palo Alto, Calif.).Briefly, 1×10⁶ cells were lysed using the lysis buffer provided in theassay kit. The lysate was stored at −20° C. and analyzed within 1 week.Enzyme activity was detected by the cleavage of the substrate,Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin (DEVD-AFC) to AFC,which emits a yellow-green fluorescence at 505 nm. The increase incaspase activity is measured as the ratio of the fluorescence intensityof a agent-treated sample (e.g., paclitaxel) to that of control cellscollected at the same time.

Externalization of phosphatidylserine. Apoptotic cells lose membranephospholipid asymmetry and expose phosphatidylserine on the outerleaflet of plasma membrane. The externalized phosphatidylserine waslabeled with Annexin V attached to a fluorescence probe FITC (greenfluorescence), using the ApoAlert Annexin V assay (Clontech). Asapoptosis progresses, propidium iodide penetrates the cell membrane andstains the cytoplasm (yellow-red stain). Briefly, cells were suspendedin binding buffer, incubated with Annexin V-FITC and propidium iodidefor 5-15 min in the dark, and examined by fluorescence microscopy. Cellslabeled with FITC and/or propidium iodide were scored.

PARP Cleavage. PARP (116,000 d) undergoes proteolytic cleavage betweenAsp 216 and Gly 217, to yield a fragment containing thecarboxyl-terminal catalytic domain (˜85,000 d), and a fragmentcontaining a truncated amino-terminal DNA-binding domain (˜26,000 d).Asp 216 is the preferred cleavage site for caspase-3 and other closelyrelated proteases (Wen L P et al. (1997) J Biol Chem 272:26056-26061;Lazebnik Y A et al. (1994) Nature 371:346-347; Cohen G M (1997) BiochemJ 326:1-16). PARP cleavage was analyzed by immunoblotting. Briefly, 10⁶cells were washed with ice-cold PBS containing protease inhibitors (1 mMphenylmethylsulfonyl fluoride and 0.5 mg/ml each of leupeptin andaprotinin), and resuspended in a reducing loading buffer (62.5 mM Tris,pH 6.8; 6 M urea; 10% glycerol; 2% sodium dodecyl sulfate (SDS); 0.003%bromophenol blue; 5% 2-mercaptoethanol). Protease inhibitors were usedto minimize proteolytic cleavage of PARP during processing. The2-mercaptoethanol solution was added (50 μl of 14.3 mmol/ml loadingbuffer) immediately prior to use. Samples were sonicated on ice,resuspended using a 21 gauge needle, and then incubated at 75° C. for 15min. After centrifugation, an aliquot representing 1.5×10⁵ cells wasloaded on a 10% SDS polyacrylamide gel and run at 30 mA overnight. Thegel was transferred onto a 0.2 mm nitrocellulose membrane byelectroblotting. The membrane was incubated with blocking solution (5%non-fat dry milk in PBS containing 0.1% Tween 20) for 1 hr, followed byincubation with the PARP monoclonal antibody overnight at 4° C. andsubsequently the anti-mouse immunoglobulin. After washing the membranein PBS twice for 5 min each and once for 30 min, the immunoreactivebands were visualized by incubation of the membrane with thechemiluminescence immunoblot kit.

Release of DNA-Histone Complex to Cytoplasm. The level of DNA-histonemono- and oligonucleosomes in the cytoplasm was measured using the celldeath detection ELISA kit. Briefly, 5000 cells were lysed in the lysisbuffer. The cytoplasmic fractions of the lysates were placed in a flaskprecoated with mouse antihistone primary antibody and mouse anti-DNAantibody conjugated to peroxidase. The peroxidase substrate,2,2′-azido-di-[3-ethylbenzthiazoline sulfonate], was applied and theabsorbance at 405 nm was measured.

DNA Fragmentation. DNA fragmentation was measured by agarose gelelectrophoresis as described in Gavrieli et al. ((1992) J Cell Biol119:493-501). Briefly, cells were incubated at 37° C. for 30 min in 10mM Tris-HCl, 100 mM EDTA (pH 8.0), 20 mg/ml RNase A, and 0.5% SDS. Thecell suspension was then treated with 200 mg/ml proteinase K at 50° C.for 16 hr. DNA was extracted twice with phenol/chloroform (1:1) and oncewith chloroform, and precipitated by adding 0.2 volume 10 M NH₄Cl and 2volumes ethanol. The pellet obtained after centrifugation wasresuspended in 100 mM Tris-HCl and 10 mM EDTA (pH 8.0). The amount ofDNA was measured by the absorbance at 260 nm, using a spectrophotometer.Samples showing a 260:280 absorbance ratio of >1.8, which ascertainedthe purity of the isolated DNA, were analyzed by gel electrophoresis.Equal amounts of DNA were loaded on a 1.5% agarose gel containing 0.5mg/ml ethidium bromide, and run at 2 V/cm for 4-5 hr inTris-acetate/EDTA electrophoresis buffer. The DNA laddering pattern wasvisualized by UV transillumination and photographed.

EXAMPLE 1 Methods for Improved Measuring of Telomerase Activity

In the previously described polymerase chain reaction (PCR)-basedtelomeric repeat amplification protocol (TRAP), an oligonucleotideprimer was first extended by telomerase, which then serves as templatesfor PCR amplification, and the telomerase activity is measured by theability of the enzyme-containing cell extract to add telomeric repeatsto the primer (Kim et al. (1994) Science 266:2011-22105). Although theoriginal TRAP assay is highly sensitive and permits the detection oftelomerase activity in small quantity of cells and tissues, itsquantitative ability is severely compromised by the poor reproducibilityand the presence of inhibitors in cell and tissue extracts, whichinhibit the PCR amplification step (Wu Y Y et al. (2000) Clinica ChimicaActa 293: 199-212). These two problems are overcome by the followingimprovements.

First, the extraction procedures in the conventional TRAP were notsufficient to completely lyse the cell nuclei and the extractionefficiency varied with the ratio between the volume of the extractionbuffer and the volume of cells. Hence, the first improvement was todevelop extraction procedures that enable complete lysis of cell nuclei,as follows. For the preparation of cell and tissue extracts, cells andtissue were harvested, washed, and stored at −70° C. Cells were thenlysed in a SDS-based lysis buffer (10 mM Tris.HCl, pH 7.5, 1 mM MgCl₂, 1mM EGTA, 0.1 mM phenylmethylsulfonyl fluoride, 5 mM β-mercaptoethanol,0.1% CHAPS, and 10% glycerol), on ice for 10 min. To this mixture, threevolumes of CHAPS-based buffer (10 mM Tris-HCl, pH 7.5, 1 mM MgCl₂, 1 mMEGTA, 0.1 mM phenylmethylsulfonyl fluoride, 5 mM P-mercaptoethanol, 0.5%CHAPS, and 10% glycerol) were added and kept on ice for 5 min. SDS is astrong detergent which can completely lyse the cell including thenucleus. For the extraction of tissues, tissues were homogenized inSDS-based lysis buffer with a Kontes pellet pestle (Fisher, Pittsburgh,Pa.), mixed with three volumes of CHAPS-based buffer, and centrifugedwere centrifuged at 8,000 g for 15 min at 4° C. The supernatant was keptat −20° C. The protein concentration of the supernatant was determinedusing Bicinchoninic Acid Kit (Sigma, St Louis, Mo.). The proteinconcentration was adjusted to 0.5 μg/μl with CHAPS-based buffer. Sampleswith equal amounts of protein (1-2 μg) were analyzed by TRAP. In thisassay, the enzyme activity is measured as the synthesis of repeatingtelomere hexamers (TTAGGG; SEQ ID NO: 10). The primers were TS(5′-AATCCGTCGAGCAGAGTT-3′ (SEQ ID NO: 1)) for telomere elongation, andTS and CX (5′-CCCTTACCCTTACCCTTACCCTAA-3′ (SEQ ID NO: 2)) for PCRamplification of telomerase product. The TRAP reaction mixture contained2-4 μl of protein extract, 20 mM Tris.HCl (pH 8.3), 1.5 mM MgCl₂, 63 mMKCl, 0.005% Tween-20, 1 mM EGTA, 50 μM dNTPs, 0.1 μg of TS primer, 0.1mg/ml bovine serum albumin. The mixture was incubated at 30° C. for 30min, then at 90° C. for 2 min.

The second problem with the original TRAP method was the presence ofinhibitors of the PCR amplification. Hence, the second improvement wasto remove the inhibitors after the primer extension step. This wasaccomplished by using phenol/chloroform extraction of the cell lysate,as follows. One μg of tRNA and 0.1 ng internal control template wereadded to the reaction solution after primer extension. The total volumeof the mixture was brought to 100 μl with water, mixed with an equalvolume of buffer saturated phenol (Life Technology) and then centrifugedat 18,000 g for 3 min, at room temperature. The top aqueous layer (90μl) was carefully transferred to a new tube and mixed with an equalvolume of chloroform: isoamyl alcohol (24:1) solution and centrifuged at18,000 g for 3 min, at room temperature. The top aqueous layer (80 μl)was again transferred to a new tube and the nucleotides wereprecipitated with 0.2 volume of 3 M sodium acetate (pH 5.3) and 3 volumeof 100% ethanol at −70° C. for at least 2 hr. The mixture wascentrifuged at 18,000 g for 15 min at 4° C. The supernatant wascarefully removed using suction. The remaining pellet was washed oncewith cold (−20° C.) 100% ethanol, centrifuged and the ethanol layer wasremoved using suction. The washed pellet was allowed to completelyair-dried and then resuspended in 20 μl 1× PCR buffer [1× PCR buffercontains 100 mM Tris-HCl (pH 8.3), 1.5 mM MgCl₂, 50 mM KCl], to which 20μl master 11 solution [which contains 1× PCR buffer, 0.2 μg CX primer(SEQ ID NO: 2)₄, 0.12 μg TS primer, 2.5 ng each of INS1 and INS2primers, 50 μM dNTP, 0.8 μl Advantage cDNA Polymerase Mix (50×)(Clontech, Palo Alto, Calif.)] was added.

The internal standard consisted of upstream primer (INS1):5′-ACACAACATACGAGCCGGAA-3′ (SEQ ID NO: 3), and downstream primer (INS2):5′-TTAATGCAGCTGGCACGACA-3′ (SEQ ID NO: 4), and amplified 130 bp ofpGEM-T Easy vector (Promega). The template for the internal standard wasamplified by PCR and purified by spin columns (QIAquick PCR purificationkit, Qiagen, Valencia, Calif.). The concentration was determined by aspectrophotometer.

PCR was performed on the GeneAmp PCR system 2400 (Perkin Elmer) andinitiated by 1 cycle at 94° C. for 3 min, 50° C. for 4 min, and 68° C.for 2 min, then 25 rounds at 94° C. for 30 sec, 50° C. for 30 sec, and68° C. for 90 sec, followed by 68° C. for 10 min. Ten μl loading buffer(0.25% bromophenol blue, 30% glycerol) was then added to the reactionand analyzed by electrophoresis in 0.5× Tris-borate-EDTA buffer on a 10%polyacrylamide nondenaturing gel. The gel was then dried and theamplification products were detected.

The detection of the amplified products was performed using bothradioactive and nonradioactive methods. The radioactive method includedthe use of 4 μCi ³²P-dCTP (ICN, Costa Mesa, Calif.) in the master IImixture, whereas the nonradioactive method used only dNTP that were notradiolabeled. For the radioactive method, the dried gel was exposed toX-ray film overnight for autoradiography. For the nonradioactive assay,the gel was stained with 0.25 μg/ml ethidium bromide in water for 20min, then washed in water for 30 min. For both methods, the image wascaptured by a gel documentation system (Gel Print 2000i, Biophotonics)and analyzed using the GPTools software package. The bands correspondingto PCR amplification products were quantified using a betascanner, andthe intensity of bands was measured.

To quantify the telomerase activity, standard curves were constructed byusing different amounts of proteins. A plot of the ratio of(amplification products of the cell or tissue extract) to (internalstandard amplification products) versus protein concentrations showed alinear increase of telomerase activity with protein concentrations(r²=0.99), indicating the linear relationship between the amount of TRAPproducts and telomerase activity. For each sample, the amount of TRAPproducts was calculated as the sum of the intensity of individual DNAladders using GPTools software. The amount of TRAP products werenormalized by the intensity of the internal control band, and used tocalculate the TRAP activity.

EXAMPLE 2 Methods for Improved Measurement of Telomere Length

Telomere length is usually reported as a mean length from allchromosomes. The standard method for measuring telomere length is bySouthern blot hybridization (Counter C M, (1996) Mut. Res. 366:45-63;Southern, E., (1975) J. Mol. Biol. 98:503-517). This is a multi-stepmethod which entails (a) cleaving purified DNA with restriction enzymes,(b) separating the DNA fragments by size on an agarose gel, (c)denaturing and transferring the fragments to a membrane (usuallynitrocellulose or nylon), (d) hybridizing the telomere with aradioactive telomere probe by immersing the membrane in aprobe-containing solution, and (e) removing the unhybridized probe bywashing the membrane. There are several disadvantages of this methodincluding (a) loss of DNA due to the multiple transfer steps, (b)labor-intensiveness of the procedure, (c) potential loss of DNAmolecules due to their inability to be immobilized on the membrane(which is a concern for short DNA molecules), (d) incompleteavailability of the immobilized DNA molecules for hybridization with thetelomere probe, (e) probe hybridization with unintended target moleculesleading to high background noise, (f) loss of signal due to theextensive washing required to reduce the background noise, and (g)limited sensitivity due to the short length of the probe (Sambrook J etal. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring HarborLaboratory Press; Gillespie D(1990) Vet. Micro., 24: 217-233). Althoughthe Southern blot is theoretically useful for both qualitative andquantitative measurements of the relative telomere amount and thetelomere length, it is usually only adequate for analysis of therelative telomere length (e.g., changes with time or due to drugtreatment) due to the problems mentioned above and is seldom used toquantify the changes in telomere amount.

These problems were overcome by the following improvements that enablethe measurement of the amount and the average length of telomeres. Inthis improved telomere amount and length assay (hereafter referred to asTALA), the probe is added to a solution containing restricted DNAfragments and allowed to hybridize with the telomeres. These hybridizedtelomeres are then separated from the unhybridized probes using gelelectrophoresis and quantified using autoradiography. As shown below,TALA is more rapid and has a higher sensitivity and reproducibility whencompared to the Southern blot analysis.

For the extraction of DNA, exponentially growing cells were treated with0.25% trypsin, scraped with a rubber policeman, collected and washed twotimes with phosphate-buffered saline (PBS). Cells (2.5×10⁶) were lysedby incubating at 37° C. for 30 min in 10 mM Tris HCl, pH 8.0, 20 mMEDTA, 0.5% SDS, 20 μg/ml RNAse A. Proteinase K (50 μg) [Sigma, St.Louis, Mo.] was then added and the mixture was incubated at 50° C.overnight. Cells were extracted with 2 volumes of phenol, 1 volume ofchloroform/isoamyl alcohol (24:1)/phenol (50:50) and 2 volumes ofchloroform/isoamyl alcohol. The genomic DNA in the supernatant wasprecipitated with 2 volumes 100% ethanol/0.1 volume of 3M sodiumacetate, washed with 70% ethanol, allowed to air dry for 30 min, anddissolved in double distilled water overnight at 4° C. The DNAconcentration was determined by A₂₆₀ using a spectrophotometer.

For the digestion of DNA, genomic DNA (10 μg) was digested withrestriction enzymes, i.e., 10 units each of HinfI, HaeIII, and HhaI inReACT 2 buffer (all from Gibco BRL), for 2 hr at 37° C. Telomere repeatswere separated from free nucleotides and small DNA fragments using theQIAquick PCR Fragment Removal Kit containing PB, PE and Elution Buffers(Qiagen Inc., Santa Clarita, Calif.). Briefly, the sample was dilutedwith PB Buffer, placed on a QIAquick spin column, washed with PE Bufferand eluted off the column with Elution Buffer (10 mM Tris HCl, pH 8.5).

The telomere probe (5′-TTAGGGTTAGGGTTAGGGTTAGGG-3′) (SEQ ID NO: 11)(Bio-Synthesis Inc., Lewisville, Tex.) was 3′ end-labeled with γ-³²P bythe forward reaction using T4 Polynucleotide Kinase (Gibco BRL), asdescribed by the manufacture. Briefly, 100 ng of unlabeled probe wasincubated with the forward reaction buffer (Gibco BRL), 50 μCi γ-³²P(ICN, Costa Mesa, Calif.) and 10 units Polynucleotide T4 Kinase for 30min at 37° C. The labeled probe was harvested using the QIAquickNucleotide Removal Kit containing PN, PE and Elution Buffers (Qiagen).Briefly, the sample was diluted with PN Buffer, placed on a QIAquickspin column and washed twice with PE Buffer, and the labeled probe waseluted off the column with Elution Buffer (10 mM Tris-HCl, pH 8.5).

For the measurement of the amount and length of telomere, the telomericDNA fragments were hybridized with the telomere probe as follows. Therestricted DNA (2.25 μg) was hybridized with 1.5 ng γ-³²P-labeled probein the ReACT 2 buffer (Gibco BRL). The restricted DNA was denatured at98° C. for six min, hybridized at 55° C. for various times ranging from0.5 to 20 hr, and further cooled to 4° C. for at least 5 min. Thedenaturation, hybridization, and cooling were performed in athermocycler (Perkin Elmer model 2400 PCR machine). The hybridizedtelomeres were separated from the unincorporated probe by running themixture on a 0.7% agarose (Gibco BRL) gel for 6 hr at 90 volts in 1× TBE(0.045 M Tris-borate, 0.001 M EDTA). The unincorporated probes wereeluted off the gel under these conditions, whereas the hybridizedtelomeres remained on the gel. The gel was then dried under vacuum for 2hr and the signal was detected by placing the dried gel on aphosphorimager screen (Molecular Dynamics, Sunnyvale, Calif.) for atleast 6 hr. The resulting autoradiograph was captured using aphosphorimager (Molecular Dynamics). The intensity of the signal wasdetermined using Imagequant (Molecular Dynamics) software. The volumereport option was used to generate the total intensity within each lane.The average background signal was defined by the signal in the areaswhere there was no telomere signal. Because the signal is composed oftelomere repeats of different lengths and appears as a long smear, themaximum telomere length was defined as the length corresponding tohighest position on the gel where the signal first exceeded three timesthe average background signal. The mean telomere length was defined asthe length corresponding to the position where 50% of the total signaloccurred. The mean telomere length was determined using molecularmarkers as references and data was analyzed by the Microsoft Excelprogram.

As a comparison, the results of TALA were compared to the results of thecommonly used Southern blot hybridization method. Southern blothybridization was performed as described (Allsopp R C et al. (1990)Proc. Natl. Acad. Sci. USA, 89:10114-10118; Harley C B et al. (1990)Nature, 345:458-460) with the following modifications. Genomic DNA(10-15 μg) was digested with restriction enzymes as described above. Thedigested DNA was separated on a 0.7% agarose (Gibco BRL) gel for 6 hr at90 volts in 1× TBE (0.045 M Tris-borate, 0.001 M EDTA), and transferredto a nylon support (MagnaCharge, MSI, Westboro, Mass.) by capillaryaction with 10×SSC (0.3 M NaCl, 0.03 M Na Citrate). After drying at 80°C. for 2 hr. the blot was incubated with a prehybridization buffer for18 hr at 50° C. This buffer consisted of 5× Denhardt's (i.e., 0.001 g/mleach of type 400 Ficoll, polyvinylpyrrolidone, fraction V bovine serumalbumin), 6×SSC, 0.5% SDS, and 100 μg/ml sonicated salmon sperm DNA(Gibco BRL). The blot was then hybridized with the γ-³²P-labeledtelomere probe for 20 hr at 50° C., washed two times at 50° C. with 0.1%SDS, 1.0×SSC and two times with 0.1% SDS, 0.1×SSC. The telomere signalwas detected as described in TALA.

The amount and length of telomere in several human cancer cells, i.e.,head and neck FaDu cells, breast MCF 7 cells, bladder RT4 cells, ovarianSKOV3 cells, lung A549 cells, and prostate PC3 cells, were analyzed byTALA. The maximum telomere lengths in these cell lines ranged from˜3,000 to ˜10,000 base pairs (bp) and the mean telomere length rangedfrom ˜1,500 to ˜3,300 bp (summarized in Table 2). Statistical analysisindicates no significant differences in the telomere lengths betweenBC19, FaDu, MCF7, and PC3 cells (p=0.081 for maximum telomere length andp=0.868 for mean telomere length, 4 determination for each cell lines),but a significantly higher telomere length in the SKOV3 cells, comparedto the other cell lines.

The results show that TALA can be used to accurately determine DNAamounts. When TALA was performed on various amounts of DNA (0.5 μg-16μg), a linear standard curve (r²=0.985) was produced over the course ofthree replicates. Likewise, the Southern blot procedure performedslightly worse than TALA in determining DNA amounts (2 μg-24 μg) overthe course of three replicates (r²=0.923). However, the standard curvesshowed that TALA is at least 4 times more sensitive than the Southernblot procedure when determining DNA amounts. TALA was able to accuratelydetect 0.5 μg of DNA, whereas the Southern blot procedure was unable todetect 2 μg but able to detect 4 μg of DNA.

To determine the time required for efficient hybridization of telomereprobes with the telomeres, the telomere signal in the SKOV3 cells wascompared after hybridization times of 0.5 to 20 hr. The SKOV3 cells wereused because they showed the longest telomere length and best intensitysignal among the five cell lines tested. The results indicate arelatively constant amount of telomere at these hybridization times. Theintensity of the telomere signal (expressed as relative gray scaleunits) was 50±8% for 0.5 hr of hybridization, 50±9% for 1 hr, 46±3% for2 hr, and 36±7% for 20 hr. The slightly lower signal (˜25%) resultingfrom the longest hybridization time of 20 hr was not significantlydifferent from the other hybridization times (p=0.122). It was concludedthat maximum hybridization between telomeres and telomere probe wasachieved in 0.5 hr.

TALA and Southern blot analyses both require DNA digestion andquantification of the ³²P signal. The major difference between the twomethods is that the TALA requires fewer transfer steps and does notrequire the prehybridization step to block the nonspecific hybridizationof the telomere probe to the membrane. Overall, the Southern blotanalysis of telomere length required ˜42 hr of sample processing andtransfer prior to quantification of the ³²P signal, whereas TALArequired only ˜11 hr. The fewer sample manipulations with TALA alsominimized the loss of sample. Further, the elimination of the membraneprehybridization and hybridization steps with TALA reduced thebackground noise and hence resulted in a higher signal-to-noise ratio(see below). Consequently, TALA was more sensitive and required lesssample compared to the Southern blot analysis.

Compared to the TALA results, the results of the Southern blot analysisshow a higher background noise, presumably due to non-specific bindingof the telomere probe with non-telomeric molecules on the membrane. Alsoobserved was a diminished telomere signal for four of the five celllines tested, even though the amount of DNA used for analysis was 4times that used in TALA. This decreased signal occurred in the four celllines with shorter telomeres (i.e., BC19, FaDu, MCF7, PC3) and resultedin an intensity signal which was less than 3 times background.Therefore, the telomere lengths from these cell lines could not bereproducibly determined (Table 2). The high background noise and thelower signal intensity contributed to a lower signal-to-noise ratio inthe Southern blot results, compared with the TALA results. The highersignal-to-noise ratio further shortened the phosphorimager exposure timerequired for the detection of the telomere signal in TALA compared tothe Southern blot analysis, i.e., 6 hr versus 24 hr.

The maximum telomere length of the SKOV3 cells determined by theSouthern blot analysis was comparable to the TALA results (Table 2).However, the mean telomere length as determined by Southern blotanalysis was ˜2 times the value determined by TALA. A comparison of thetelomere signal indicates that the ³²P signal in the TALA resultsextended from 2907 to 3703 bp whereas the signal in the Southern blotresults extended from 6924 to 9760 bp. This is because of the SouthernBlot results showed a signal-to-noise ratio that was below the detectionlimits. The omission of the signal corresponding to the shortertelomeres resulted in the erroneously higher mean telomere lengthobserved in the Southern blot.

The reproducibility of the telomere length determination by TALA andSouthern blot analysis was compared. For TALA, the telomere length infive cell lines were measured in 4 different experiments. Southern blotanalysis was limited to SKOV3 cells (n=3 experiments), because the othercell lines which contained shorter telomeres did not give reproducibleresults (see above). Data in Table 2 show comparable coefficients ofvariation for the maximum and mean telomere lengths in the SKOV3 cells,determined using the two methods (i.e. 12-15% for TALA and 17-19% forSouthern).

The reproducibility of the telomere amount determination by TALA andSouthern blot analysis was also compared. For TALA, the telomere amountin three cell lines were measured in 3 different experiments, each withfive replicates. The average coefficient of variation were 16% for BC19cells, 14% for MCF7 cells, and 9% for SKOV3 cells, with an overallaverage value of 13%. Southern blot analysis was limited to SKOV3 cells,for the reason stated above. The coefficient of variation of theSouthern blot results obtained from 3 different experiments, each withfour replicates of SKOV3 cells, ranged from 16% to 30%, with an overallaverage of 23%. A comparison of the coefficients of variation for theSKOV3 cells data indicate a higher reproducibility for TALA than forSouthern blot analysis. TABLE 2 Determination of telomere length by TALAand Southern blot analysis. The maximum and mean (centroid) telomerelengths (the length of the terminal restriction fragment) weredetermined using TALA or Southern blot analysis. Data are reported asmean∀standard deviation (n = 4), with coefficients of variation inparenthesis. TALA Southern Blot Maximum Mean Maximum Mean Cell LineLength, bp Length, bp Length, bp Length, bp BC19 2779∀217 1514∀127 Notdetermined Not determined  (7.8%) (8.4%) FaDu 3282∀466 1802∀171 Notdetermined Not determined (14.2%) (9.5%) MCF7 3366∀253 1700∀98 Notdetermined Not determined  (7.5%) (5.8%) PC3 3261∀258 1777∀118 Notdetermined Not determined  (7.9%) (6.6%) SKOV 3 9660∀1449 3305∀39813,099∀2542 8342∀1418 (15.0%)  (12%) (19.4%) (17.0%)

EXAMPLE 3 In Vitro Demonstration that Treatment with Cytotoxic AgentsReduces Telomere Length

This example demonstrates that treatment with cytotoxic agents resultsin shortening and/or deletion of telomere.

Human ovarian SKOV cancer cells and human pharynx FaDu cancer cells weretreated with paclitaxel. Cells detached from the growth surface wereharvested by transferring the cell-containing culture medium to a secondculture flask. Cells that remained attached to the growth surface wereharvested using trypsinization. Detached and attached cells wereprocessed for telomere length measurements. Two methods were used toevaluate the effect of cytotoxic treatment on telomere. The first methodused TALA to measure the average amount and length of telomere. Thesecond method used FISH to detect the presence or absence of telomereand the length of telomere (by monitoring the fluorescence intensity ofthe telomere-binding probes). FISH analysis detects the telomere statusof individual chromosomes in individual cells and provides asemi-quantitative measurement of telomere shortening (i.e. partial orcomplete deletion of telomeres). TALA measures the average telomerelength in all cells, and the TALA results represent a populationaverage, and cannot distinguish if all cells have the same extent ofshortening or if some cells have a greater extent of shortening (i.e.10% shortening in 100% cells or 10% cells with 100% shortening).

The TALA results show that treatment of SKOV3 cells with 200 nMpaclitaxel for 24 hr resulted in a 13% shortening of telomeric length,compared to untreated controls. Extending the paclitaxel treatment to 36hr did not produce a greater telomere shortening. The doubling time forSKOV3 cells was 25 hr. This extent of telomere length shortening is muchgreater than the shortening due to aging or telomerase inhibition (i.e.average of <1% per doubling) reported for other cells, and is identicalto the shortening that results in the death of HeLa cells afterinhibiting its telomerase for 23-26 doublings (Feng et al. (1995)Science 269:1236-1241).

The FISH results showed that after treatment with 200 nM paclitaxel,some of the FaDu and SKOV3 chromosomes showed lower or no fluorescencesignals, compared to the untreated control cells. This data indicates ashortening or deletion of telomeres. This effect was detectable after 1hr paclitaxel treatment, and increased with treatment. For SKOV3 cells,40% and 90% of the detached cells showed damaged telomeres aftertreatment with paclitaxel for 1 and 12 hr, respectively, whereas <5% ofthe attached cells showed damaged telomeres at these time points. Thisdata indicates a significant difference in the telomere status in theattached and detached cells after paclitaxel treatment.

EXAMPLE 4 Agent-Induced Telomere Damage Occurs Prior to Other HallmarkApoptotic Changes

This examples demonstrates that cytotoxic treatments cause damage totelomere in cells and that telomere damage occurs before other changesin the apoptotic cascade. Hence, damages to telomere appears to be aninitiator of apoptosis, rather than a consequence of the apoptoticprocess.

Apoptosis is associated with morphological changes, including membraneblebbing, cellular shrinkage, chromatin condensation, and detachmentfrom the extracellular matrix (Kerr J F R et al. (1980) Br J Cancer26:239-257). It is also associated with biochemical changes, includingactivation of a cascade of proteases such as the caspases andendonucleases, cleavage of poly-ADP-ribose polymerase (PARP), andeventually fragmentation of genomic DNA (Patel T et al. (996) FASEB J10:587-597; Arends M J et al. (1990) Am J Pathol 36:593-608; Wyllie A H(1980) Nature 284:555-556). Caspase 3 is considered the first caspaseinvolved in the execution phase of apoptosis and is activated by theproteins involved in the initiating phase (i.e., caspase 8, caspase 9,and cytochrome C). Caspase 3 cleaves target proteins including PARP,gelsolin, p21-activated kinase 2, and DNA fragmentation factor (NeamatiN. et al. (1995) J Immunol 154:3788-3795; Lazebnik Y. A. et al. (1995)Proc Natl Acad Sci USA 92:9042-9046; Voelkel-Johnson C. et al. (1995) JImmunol 153:4247-4255; Wen L. P. et al. (1997) J Biol Chem272:26056-26061; Kaufmann S. H. et al. (1993) Cancer Res 53:3976-3985;Kothakota S. et al. (1997) Science 278:294-298; Rudel T. and Bokoch G.M. (1997) Science 276:1571-1574; Liu X. et al. (1997) Cell 89:175-184).PARP is an essential DNA repair enzyme. Cleavage of PARP prevents DNArepair, activates a calcium/magnesium-dependent endonuclease, andresults in internucleosomal DNA fragmentation. Cleavage of DNAfragmentation factor is also associated with internucleosomal DNAfragmentation. Internucleosomal DNA fragmentation, where the nuclear DNAis sequentially degraded to 300 kb, 50 kb, and ˜185 bp fragments, is alate event in apoptosis that is frequently used to confirm apoptoticdeath (Collins J. A. et al. (1997) J Histol Cytometry 45:923-934). Thefragmented DNA is released into the cytoplasm as a DNA-histone complex.Another hallmark apoptotic change is the loss of cell membranephospholipid asymmetry where phosphatidylserine is externalized on theouter leaflet of plasma membrane.

Studies were performed to establish the timing of the biochemicalchanges in paclitaxel-induced apoptosis in human prostate PC3 cancercells and human pharynx FaDu cancer cells. In both cells, paclitaxeltreatment (20 nM in PC3 cells and 200 nM in FaDu cells) resulted in celldetachment from the growth surface; the detached cells, as a fraction ofthe total cells, increased with treatment duration. As shown in Example3 and later in this Example, the attached and detached cells showedqualitatively and quantitatively different characteristics. For example,telomere damage was frequent (up to 90%) and several biochemical changestypical of apoptosis were detected in the detached cells, whereastelomere damage was infrequent (<5%) and not all of the hallmarkapoptotic changes were detectable in the attached cells. Thesimultaneous presentation of telomere damage and several apoptoticchanges in the detached cells and the simultaneous absence of thesechanges in the attached cells suggest a link between the agent-inducedtelomere damage and apoptosis.

For PC3 cells, the detached cell fraction reached 68% at the end of the96-hr experiment, whereas the untreated control samples showed <1%detachment. For FaDu cells, the detached cell fraction was about 40% atthe end of the 48-hr treatment. For both cells, the attached anddetached paclitaxel-treated cells showed different biochemicalproperties. For PC3 cells, the detached cells exhibited the fullspectrum of apoptotic changes including activation of caspase 3, PARPcleavage, DNA fragmentation, and release of DNA-histone complex to thecytoplasm, whereas the attached cells only showed activation ofcaspase-3-like proteases but not the remaining apoptotic changes.Activation of caspases in the attached cells was several-fold lower andoccurred at a later time (i.e., 24 versus 12 hr) compared to thedetached cells. In the detached cells, caspase activation was firstdetected at 12 hr and peaked at 36 hr, whereas PARP cleavage was firstdetected at 24 hr and was completed prior to 72 hr. In contrast, theextent of internucleosomal DNA fragmentation and the release ofDNA-histone complex to the cytoplasm (both were first detected at 24 hr)were cumulative over time up to the last time point of 96 hr.

For FaDu cells, the fraction of the paclitaxel-treated cells thatremained attached to the growth surface and was labeled for theexternalized phosphatidylserine was nearly identical to that in theuntreated controls (i.e., between 6-8%), whereas the paclitaxel-treatedcells that were detached from the growth surface showed time-dependentincreases with the labeled fraction becoming significantly higher thanthe value in the untreated controls at 12 hr and reaching 60% at 24 hr.Among the paclitaxel-treated FaDu cells, only the detached and not theattached cells, showed cytoplasmic DNA-histone complex and DNAladdering.

A comparison of the results in Example 3 and this example on the timecourse of paclitaxel-induced damage to telomere and apoptotic changesindicates that telomere damage (first detected at 1 hr) was detectedprior to other apoptotic changes such as caspase activation andphosphatidylserine externalization (both of which were first detected at12 hr), PARP cleavage, release of DNA-histone complex into the cytoplasmand DNA laddering (all three of which were first detected after 24 hr).Taken together, these data in PC3 and FaDu indicate paclitaxel-inducedtelomere damage occurred prior to other hallmark apoptotic changes.

EXAMPLE 5 Cytotoxic Treatments Cause a Transient Increase in TelomeraseActivity in Cells and Solid Tumors

This examples demonstrates that treatment of tumor cells with cytotoxicagents with different mechanisms (e.g., paclitaxel, cisplatin,radiation) increases telomerase activity (up to 100%) in multiple humancancer cells, including prostate PC3, pharynx FaDu, bladder RT4 cells,and in solid tumors (FaDu) maintained in immunodeficient mice.

FIG. 1 shows the effect of paclitaxel on the telomerase activity of FaDucells. Samples were taken at various time points during paclitaxel (200nM) treatment and analyzed for telomerase activity. Paclitaxel treatmentinduced a transient increase in telomerase activity, which peaked at˜180% of control value at the first time point of 3 hr, returning to thebaseline level at 24 hr, followed by a gradual decrease to 50% of thecontrol values at 48 hr. Similar results were found for other cytotoxictreatments, including treatments with cisplatin, radiation,hyperthermia, and serum starvation. For cisplatin and radiation, peaktelomerase activity was found at between 6 to 12 hr and returned to thebaseline/control level at 24 hr, followed by a decrease to 50% of thebaseline level at 48 to 96 hr.

A second study evaluated whether the transient induction of telomeraseactivity by cytotoxic treatment also occurs for cells maintained assolid tumors in animals. This study was performed using the FaDuxenograft tumor maintained in immunodeficient mice. Five tumors obtainedfrom five mice were processed and maintained as histocultures.Histocultures are tumor fragments that are maintained on collagen gel.Tumor histocultures were treated with 1 μM paclitaxel for differentdurations. The paclitaxel concentration of 1 μM was chosen because it isclinically achievable and caused 50% cytotoxicity in solid tumors (Ganet al (1996) Cancer Res 56:2086-2093). FIG. 1 shows that paclitaxeltreatment induced telomerase activity in the FaDu solid tumors; theenzyme activity peaked at 12 hr, returned to baseline level at between24 to 48 hr and further declined to about 50% of the baseline level at96 hr.

EXAMPLE 6 Presence of DNA Free Ends Induces Telomerase Activity

This example demonstrates that the presence of DNA free ends, e.g., DNAwith damaged and uncapped telomere, induces telomerase activity.

That the presence of DNA free ends induces telomerase activity wasconfirmed by studying the effect of transfecting cells with linearizedplasmid DNA without telomeres. Because the primary function oftelomerase is to synthesize telomere repeats to stabilize chromosomalends following DNA replication (using double stranded DNA), the studyused double stranded DNA. Briefly, the pGL3-control plasmid DNA (5,268bp) was treated using the restriction enzyme SmaI which cuts thecircular DNA into a linearized DNA fragment with two free endsunprotected by telomeres. The fragments were then randomly labeled withfluorescein-labeled dNTPs, and the transfected cells containing thelabeled DNA fragments were detected by flow cytometry and separated fromthe non-transfected cells by cell sorting. The telomerase activity innontransfected and transfected cells was compared to determine theeffect of the presence of DNA free ends. The procedures are outlinedbelow.

The pGL3-control plasmid was obtained from E. Coli competent cells,purified using the Qiagen maxi kit, and quantified by UV absorbance at260 nm. Two hundred μg of plasmid DNA was incubated with 40 units ofSmaI (Gibco BRL) and 100 μl of 10_(x) REACT 4 buffer at 30° C. for 2 hr.Afterwards, the restriction enzyme was removed by centrifugation using aMillipore UltraFree-MC tube. The plasmid was then labeled withfluorescein-12-dUTP using a nick translation kit (Boehringer-Mannheim)and fluorescein-12-dUTP. Briefly, fluorescein-12-dUTP was incubated withdNTPs, nick translation buffer, DNA polymerase-I, and plasmid DNA at 15°C. for 90 min. The reaction was stopped by heating in a water bath at65° C. for 10 min, and the plasmid was obtained by precipitation using100% ethanol and 3 M sodium acetate.

FaDu cells were transfected with the fluorescein-labeled plasmid DNAusing Lipofectin (Gibco). Four μg of plasmid was added to 100 μl serumfree media (Opti-Mem), and then incubated with 105 μl of a premixedsolution of Lipofectin/Opti-Mem (5:100) for 15 min at room temperature.Afterwards, 795 μl of Opti-Mem was added, and the mixture was addeddrop-wise to the culture flask containing cells in subconfluent cultures(˜5×10⁵ cells). At preselected times, cells were washed and harvested,and processed for sorting.

Flow cytometric cell sorting was performed on a Coulter EPICS Elite ESPCytometer equipped with a air-cooled Argon laser. The fluoresceinexcitation wavelength was 488 nm. Optical laser alignment calibration ofthe flow cytometer was performed using Coulter's DNA-Check EPICSalignment fluorosphere beads with coefficient of variations of less than2%. Cellular debris and doublets were eliminated by forward angled and90μ light scatter. The fluorescence light emission was reflected through550 nm dichroic long pass filter and collected through a 525 nm bandpass filter. Background gating for positive transfectant cells did notexceed 1%. Viable cells were sorted at 1 droplet with a data rate of3,000-5,000 events/sec using a 100 μm flow sense quartz tip with afrequency of 32 KHz and coincidence abort activated. Sorted cells weredeflected through a 3000 V ceramic deflection sort plate and collectedinto sterile polypropylene tubes containing phosphate-buffered salinewith 1% fetal calf serum maintained at 4° C. using a temperatureregulation module. The fluorescein signal was measured in logarithmicmode. The purity of the sorted cells was greater than 96-98%.

FIG. 2 shows the changes in telomerase activity in transfected andnontransfected cells with time, after the transfection procedures. Theresults are presented as ratios to the enzyme activity in cells prior tobeing subjected to the transfection procedures. The nontransfected cells(i.e., cells that were subjected to the same transfection procedures asthe transfected cells but did not incorporate the plasmid) showed aconstant, 6-fold increase in telomerase activity at 1 and 3 hr aftertransfection, indicating that the transfection procedures inducedtelomerase. In contrast, the cells transfected with plasmid containingfree DNA ends showed a 133-fold increase at 1 hr and a 16-fold increaseat 3 hr. These results indicate that the presence of DNA free endsinduces telomerase activity and that the enzyme induction was immediateand transient.

EXAMPLE 7 Telomerase Inhibitors Enhance the Activity of Agents thatDecrease Telomere Length: hRT Antisense as the Telomerase Inhibitor

Results of Examples 3-6 suggest that cytotoxic agents induce damage totelomere, an event which precedes the earliest know hallmark event ofapoptosis as well as a transient increase in telomerase activity.Moreover, the results of Example 6 show that telomerase activity isinduced by the presence of DNA free ends. These results suggest that thetransient telomerase induction is a mechanism of protecting cells fromthe damages caused by the cytotoxic treatment. Accordingly, telomeraseinhibitors, by inhibiting the telomerase-mediated protection, canenhance the activity of cytotoxic agents. This example demonstrates thattelomerase inhibitors enhance the cytotoxicity of agents that causedamage to telomere. Two types of inhibitors were studied. They areantisense to telomerase and reverse transcriptase inhibitors, AZT andd4T. This Example describes the results of the study using the antisensemolecule. Example 8 describes the results of the study using the reversetranscriptase inhibitors.

The antisense study consisted of the following steps: (a) constructionof a sense and antisense to the RNA portion of the human telomerase(hTR), (b) stable transfection of cells with the hTR antisense (or hTRsense control), and (c) determination of the ability of the hTRantisense to enhance the activity of the cytotoxic agents.

Antisense and sense constructs. The sense and anti-sense expressionplasmids for human RNA portion of telomerase were prepared by thefollowing steps: (a) obtaining a hTR segment from tumors or tumor celllines using RT-PCR, (b) using the RT-PCR product to construct arecombinant plasmid pGEM-T Easy-hTR DNA, (c) confirming the sequence andthe direction of the candidate hTR segment in the recombinant plasmid,and (d) construction of recombinant expression vectors containing eithersense or anti-sense hTR.

Briefly, total RNA was isolated from fresh bladder tumors as describedin protocol of High Pure RNA Isolation Kit (Boehringer Mannheim). RT-PCRwas performed according to the protocol of 1^(st) Strand cDNA SynthesisKit (Boehringer Mannheim). The primers for hTR are: 5′-CAG CTG ACA TTTTTT GTT TGC TCT A-3′ (SEQ ID NO: 5) and 5′-GGG TTG CGG AGG GTG GGC CT-3′(SEQ ID NO: 6). The band eluting at the position as the hTR fragment(185 bp) was excised and purified as described in the protocol ofAgarose Gel DNA Extraction Kit (Boehringer Mannheim). The purified bandwas used as the template to perform a second round of RT-PCR, whichproduct was further purified using the QIAquick PCR Purification kit(Qiagen). The PCR product was ligated with the pGEM-T Easy Vector, byincubating the pGEM-T and pGEM-T Easy Vector System (Promega) in a 10 mlreaction mixture containing 1× DNA ligation buffer, 1 ml purified hTRPCR product, 50 ng pGEM-T Easy vector and 3 units T₄ DNA ligase, at 4°C. overnight. The ligated product was transformed into the XL₁-bluecompetent cells. The transformed culture was used to prepare plasmid DNAas described in QIAprep Miniprep Kit (Qiagen). Ten clones were obtained.Sequencing was performed using pUC/M13 forward sequencing primer (5′-GTTTTC CCA GTC ACG AC-3′(SEQ ID NO: 9)) according to the fmol^(R) DNASequencing system (Promega). The sequencing results showed that all 10clones displayed the correct sequence of hTR.

The PGEM-T Easy plasmid containing the 185 bp hTR DNA fragment wasdigested by NotI. The resulting 219 bp Notl DNA fragment was purifiedfrom an agarose gel (Agarose Gel DNA Extraction Kit, BoehringerMannheim). This fragment was inserted into the NotI site of theexpression vector pOPRSVI/MCS (LacSwitch™ II Inducible MammalianExpression System). These procedures resulted in 5 clones that containedthe hTR fragment. Sequence analysis was performed using fmol^(R) DNASequencing system (Promega). The primers were the T7 promoter primer:5′-TAA TAC GAC TCA CTA TAG GG-3′ (SEQ ID NO: 7) and the T3 promoterprimer: 5′-ATT AAC CCT CAC TAA AGG GA-3′ (SEQ ID NO: 8) respectively.The result showed that one clone was sense, whereas the other 4 cloneswere antisense. These expression plasmids, designated pOPRSVI/MCS hTRsense and pOPRSVI/MCS-hTR antisense, were then transfected into humanpharynx FaDu cancer cells.

Transfection procedures. Transfection of the antisense construct used anIPTG-inducible mammalian expression system, i.e., the LacSwitch IIsystem (Stratagene), which consists of a recombinant vector (i.e.,POPRSVI) and an operator vector (i.e., pCMVLacI). The pOPRSVI vectorcontains both ampicillin and G418r resistance genes which facilitate theselection of positive clones from E. Coli and eukaryotic cells. The 8unique cloning sites of the vector facilitate bi-directional insertionof a cDNA to produce either sense or antisense transcripts. The Lacrepressor vector (pCMVLacI) produces the Lac repressor protein, whichbinds to and inhibits the operator sequence in the pOPRSVI vector andthereby inhibits the expression of the inserted gene. IPTG decreases thebinding affinity of the Lac repressor protein to the operator sequenceand triggers transcription and expression of the inserted gene. Thetransfection procedures consisted of stable transfection of FaDu cellswith first, the pCMVLacI vector, and then second, the pOPRSVI/MCS vectorwhich contained an antisense or sense hTR fragment. Transfection wasperformed using Lipofectamine-mediated gene transfer. The selection ofthe clones expressing the pCMVLacI repressor construct was performedusing 200 mg/ml of the antibiotic hygromycin. The selection of theclones expressing the pOPRSVI vector using G418. The resulting cloneswere used for experiments. IPTG was used as the inducer of the hTRantisense.

Effect of the hTR antisense on cell growth. Table 3 summarizes theresults which show a slower growth rate for the antisense+IPTG cells(i.e., cells that were transfected by hTR antisense and treated withIPTG to induce the expression of hTR) compared to cells that had eithernot been transfected with the antisense, not transfected but treatedwith IPTG, transfected with the sense and treated with IPTG, ortransfected with the antisense but without the IPTG induction (i.e.,control, +IPTG, +sense+IPTG, and antisense).

Effect of hTR antisense on the cytotoxic effect of paclitaxel. Twoclones of cells that were stably transfected with the hTR antisense werestudied. The cytotoxic effect of paclitaxel was quantified using the SRBmethod which measures the total cellular proteins. The cells transfectedwith hTR antisense were treated with IPTG for 44 (clone#1) to 57 (clone#2) days, and then with paclitaxel for 96 hours. The results, summarizedin Table 3, show that the hTR antisense enhances the paclitaxelcytotoxicity in both clones by about 2-fold, as indicated by the reducedIC₅₀ of paclitaxel in the antisense-transfected cells compared to theother control cells. TABLE 3 Effect of hTR antisense on cell growth,paclitaxel cytotoxicity, telomere length, and telomerase activity.+anti- +sense + +anti- sense + Effects Control +IPTG IPTG sense IPTGDoubling time, hr 22 23 23 23 27 IC₅₀ of paclitaxel, nM, 2.04 2.42 2.562.46 1.30 clone #1 IC₅₀ of paclitaxel, nM, 2.05 2.53 2.21 2.87 1.33clone #2 Terminal restriction 2.73 2.91 2.72 2.89 1.75 fragment, kbTelomerase activity, 100 98.9 98.2 96.5 27% % of control

Effect of hTR antisense on telomere length and telomerase activity.Telomere length was measured using the TALA method as described inExample 2. Telomerase activity was measured using the improved TRAPmethod as described in Example 1. The results, summarized in Table 3,show that the hTR antisense reduced the telomere length and telomeraseactivity.

Taken together, these results suggest that the treatment of a humancancer cell with a hTR antisense results in an inhibition of telomeraseactivity, a loss of telomeres, inhibition of cell growth, andenhancement of paclitaxel cytotoxicity.

EXAMPLE 8 Telomerase Inhibitors Enhance the Activity of Agents thatDecrease Telomere Length: AZT and D4T as the Telomerase Inhibitors

Reverse transcriptase inhibitors. Two reverse transcriptase inhibitors,i.e., AZT and d4T, were studied in multiple human cancer cell lines,including breast MCF-7 cells, pharynx FaDu cells, prostate PC3 cells,and ovarian SKOV3 cells.

Treatment Protocol. Treatment with the above agents was initiated aftercells were allowed to attach to the growth surface in culture flasks. Onthe day of experiments, the culture medium was removed and replaced withdrug-containing medium. Treatment with AZT or d4T was initiated 24 hrprior to paclitaxel treatment. This was to allow for the conversion ofthe nucleosides to nucleotides, which are the active metabolites thatinhibit reverse transcriptases including, e.g., telomerase. Afterwards,the medium was again removed and replaced with fresh media containingAZT or d4T, paclitaxel (200 nM) or a combination of paclitaxel and AZTor d4T for a treatment time of 0 to 48 hr. The AZT concentration wasselected such that this concentration, for a 72 hr treatment, produced50% reduction in total cell number, but did not cause appreciabledetachment of cells from the growth surface (i.e., <10%) nor apoptosisthat was significantly higher than in the untreated controls. Theselected AZT concentration was 10 μM for FaDu and PC3 cells, 5 μM forMCF7 cells, and 100 μM for SKOV-3 cells. For d4T, two concentrations, 20μM and 40 μM were used. At these concentrations, d4T did not result insignificant detachment of cells from the growth surface (<8% detachment,see below).

Drug activity evaluation. Agent effect was measured in three ways, i.e.,enumeration of the number of cells that remained attached in cultureflask, enumeration of the number of cells that detached from the growthsurface, and by measuring changes in apoptosis. The first measurementrepresents the overall agent effect, i.e., the combination of cytostaticand cytotoxic effects, whereas the latter two measurements reflect thecytotoxic effect. The remaining cell number after agent treatment wasmeasured using the sulforhodamine B (SRB) assay. The drug-induceddetachment of cells from the growth surface was determined by countingthe attached and detached cells. Drug-induced apoptosis was measuredusing the Cell Death Detection ELISA kit which measures the release ofDNA-histone complex from the nucleus to the cytoplasm. Detailedprocedures for these three measurements were as described underExperimental Protocols.

Among the three measurements of agent effects, the SRB measurements,because they readily provided the measurement of IC values such as IC₅₀(i.e., agent concentration required to produce 50% inhibition), can beused to evaluate the nature of agent interaction. This was performedusing two methods.

The first method used fixed concentrations of the telomerase inhibitoryagent together with increasing concentrations of paclitaxel, i.e., fixedconcentration method. The advantage of this method is that it yields theconventional sigmoidal concentration-effect curves showing increases ineffect as a function of increasing paclitaxel concentration and providesa measure of the enhancement of the paclitaxel activity at a fixedconcentration of the telomerase inhibitor. The latter method facilitatesthe selection of the dosage of the telomerase inhibitor to be usedduring in vivo studies. However, the experimental design of the fixedconcentration ratio is such that only limited concentrations of thetelomerase inhibitory agent can be studied.

The second method used fixed concentration ratios of paclitaxel and thetelomerase inhibitor, i.e., fixed ratio method. The advantage of thissecond method is that it enables the measurement of the nature of theinteraction between paclitaxel and the telomerase inhibitor at muchbroader concentration ranges as compared to the first method. Anadditional advantage is that it allows the identification of the optimalratios of the two drugs that will give the maximal synergy. For thefixed concentration method, the AZT concentrations were kept constantwhile the paclitaxel concentrations were varied between 1 nM to 10,000nM. For the fixed ratio method, the concentrations of the two drugs weremaintained at four fixed ratios of their respective IC₅₀ values (i.e.,80:20, 60:40, 40:60, 20:80). For example, a constant ratio of paclitaxelto AZT of 80:20 would represent a combination containing a concentrationof paclitaxel equal to the multiples (e.g., 0.25, 0.5, 1, 2, 3 and 4times) of the 80% value of the IC₅₀ of paclitaxel with a concentrationof AZT equal to the multiples of the 20% value of the IC₅₀ of AZT. Forthe fixed ratio method, the concentration range was between 5 to 300 μMfor AZT and 2.5 to 160 nM for paclitaxel.

Analysis of pharmacodynamic data. The drug concentration-effect dataobtained from the SRB assay of drug concentration and effect wasanalyzed by computer fitting the experimental data to an effect model asdescribed previously (Au et al. (1998) Cancer Res 58:2141-2148) usingnonlinear least square regression (NLIN; SAS, Cary, N.C.). The nature ofthe interaction between paclitaxel and AZT was analyzed by theisobologram method (Berenbaum M C (1989) Pharmacol Rev 1989:93-141).Concentration-effect curves generated for both drugs and theircombinations were used to determine the concentration of each compound,either alone or in combination needed to achieve a given level ofeffect. The combination index was calculated as follows.${{Combination}\quad{Index}} = {\frac{{IC}_{A,B}}{{IC}_{A}} + \frac{{IC}_{B,A}}{{IC}_{B}}}$where IC_(A) and IC_(B) are the concentrations of agents A and B neededto produce a given level of cytotoxicity when used alone, whereasIC_(A,B) and IC_(B,A) are there concentrations needed to produce thesame effect when used in combination. In the isobologram analysis, thecombination indices are plotted against the effect levels, in order todetermine the nature of interaction at the different effect levels. Forexample, depending on the mechanisms of drug interaction, a combinationmay give synergy at a high effect level but antagonism at a low effectlevel. A combination index value of 1 indicates additive interaction,values less than 1 indicate synergistic action, and values greater than1 indicate antagonistic interaction. Hence, a plot showing combinationindices consistently below the value of 1 at all effect levels indicatessynergistic interaction at all effect levels. Conversely, a plot showingcombination indices consistently above the value of 1 at all effectlevels indicates antagonistic interaction at all effect levels.

Effect of telomerase inhibitors on paclitaxel activity: SRB results.This study was performed in human head and neck FaDu cells. Cells weretreated with AZT for 48 hr and paclitaxel for 24 hr; AZT treatment wasinitiated 24 hr prior to paclitaxel treatment. FIG. 3 shows the resultsobtained using the fixed concentration method. Paclitaxel alone reducedthe cell number to about 65% of the control value ( i.e., a maximumeffect of 35%) at 100 nM, with no increase in drug effect when theconcentration was increased 100-fold to 10 μM. AZT enhanced thepaclitaxel effect, and the concentration-effect curve was shifted to theleft and the maximum effect was increased from 35% to 60% at 20 μM AZT.

FIG. 4 shows the results of the fixed ratio method. For this method,cells were treated with AZT for 72 hr and paclitaxel for 48 hr; AZTtreatment was initiated 24 hr prior to paclitaxel treatment. The IC₅₀equivalents on the x-axis represent the concentrations of the twocompounds required to produce 50% effect. Hence, a reduction of the IC₅₀equivalent for each of the two compounds when used in combination tobelow the value of 1 indicates an enhancement of drug effect. Theresults are similar to the results obtained with the fixed concentrationmethod; addition of AZT to paclitaxel enhanced the activity ofpaclitaxel such that the IC₅₀ equivalents for both compounds werereduced. Combinations of AZT and paclitaxel resulted in reduction oftheir respectively IC₅₀ equivalents, indicating enhancement of theagent's effect. Analysis of these results by the isobologram methodshows lower combination indices for the two combinations where theAZT:paclitaxel concentration ratios were 20:80 and 40:60 compared to thetwo combinations where the ratios were 60:40 and 80:20 (FIG. 5).Furthermore, the 20:80 and 40:60 AZT:paclitaxel combinations showcombination indices of below 1, indicating synergistic interaction, atall effect levels. The extent of synergy, which equals to the inverse ofthe combination index values, was between 3.3- to 5.1-fold for the 20:80combination and between 2.8- to 3.3-fold for the 40:60 combination. Incontrast, the 60:40 and 80:20 AZT:paclitaxel combinations showsynergistic interaction only at the higher effect levels (i.e., 340%effect for the 60:40 combination and 370% for the 80:20 combination),and antagonistic interaction (indicated by combination indices higherthan 1) at the lower effect levels.

Taken together, these SRB results indicate synergistic interactionbetween paclitaxel and AZT, but that the synergy occurs only when AZT ispresent at concentrations that are near or below the concentration thatcaused 50% reduction in total cell number.

Effect of telomerase inhibitors on Paclitaxel activity: results ondetachment of cells from the growth surface. The effect of AZT wasstudied in FaDu, MCF7, PC3, and SKOV3 cells. The results are shown inFIG. 6. In all four cell lines, the maximum number of detached cells, asa fraction of the total cell number, was <3% for the untreated controls,<10% for the AZT-treated groups, ranged between 10 to 40% in thepaclitaxel-treated groups, and increased to 40 to 90% for theAZT/paclitaxel combination groups. In all four cell lines, theenhancement of the detached cell fraction by the addition of AZT wasstatistically significant.

The effect of d4T was studied in FaDu cells. The results are similar tothose obtained with AZT; addition of d4T to paclitaxel significantlyenhanced the detached cell fraction (FIG. 7).

Effect of telomerase inhibitors on Paclitaxel activity: apoptosisresults. The effect of AZT on paclitaxel-induced apoptosis was studiedin FaDu, MCF7 and PC3 cells. The results are summarized in FIG. 8. Forall three cells, the untreated controls showed negligible release ofDNA-histone complex from the nucleus to the cytoplasm. The AZT-treatedgroups showed a slight enhancement whereas the paclitaxel-treated groupsshowed a significant enhancement. Addition of AZT to paclitaxel furtherenhanced the release of the DNA-histone complex by up to 4-fold. Thesedata indicate that AZT enhances the apoptotic effect of paclitaxel.

Effect of telomerase inhibitors on telomere length. The effect ofpaclitaxel and AZT, alone or in combination, on telomere length wasstudied using TALA. The results indicate that AZT alone did not reducethe telomere length, whereas paclitaxel treatment at 200 nM for 24 hrcaused a 13% shortening of the average telomeric length. Pretreatment ofcells with 10 μM AZT for 24 hr followed by treatment with AZT plus 200nM paclitaxel for an additional 24 hr caused a greater shortening oftelomeric length (i.e., 22% decrease). Extending the paclitaxeltreatment to 36 hr did not produce a greater telomere shortening,whereas 36-hr treatment with the two-drug combination further reducedthe telomere length (i.e., a 28% decrease). This extent of telomerelength shortening is much greater than the shortening due to aging ortelomerase inhibition alone.

The results of Examples 7 and this Example, when taken together,indicate that the telomerase inhibitors including the hTR antisense andthe reverse transcriptase inhibitors reduce telomerase activity, enhancethe shortening and/or deletion of telomere caused by cytotoxic agents,e.g., paclitaxel, and enhance the activity of the cytotoxic agents thatcause damage to the telomere. These findings indicate a new therapeuticparadigm of combining telomerase inhibitors with cytotoxic agents thatcause damage to telomeres.

EXAMPLE 9 Telomerase Inhibitors Enhance the in vivo Antitumor Effect ofAgents that Decrease Telomere Length

This example describes the enhancement of the antitumor effect of anagent that damages telomeres (i.e., paclitaxel), by the telomeraseinhibitor AZT, in immunodeficient mice bearing human head and neckcancer FaDu xenografts.

The activity of paclitaxel, with or without AZT, was evaluated inimmunodeficient mice (male Balb/c nu/nu mice, 6-8 weeks old) bearing thehuman pharynx FaDu xenografts. Xenografts were formed by subcutaneousinjection of 10⁶ viable tumor cells in 0.1 ml physiologic saline in theright and left flank areas, and were allowed to grow for about 14 daysto reach a size of >15 mm³ before drug treatment was started. The fourtreatment groups are: saline control, AZT, paclitaxel, paclitaxel+AZT.The saline control group received injections of 200 μl/day ofphysiological saline of five consecutive days. The paclitaxel groupreceived injections of 10 mg/kg/day paclitaxel dissolved in Cremophorand ethanol (i.e., Taxol) in a volume of 200 μl for five consecutivedays. The AZT group received a seven-day infusion of AZT at a rate of200 ng/hr by a subcutaneously implanted Alzet minipump. Thepaclitaxel+AZT group received the combined treatment of the paclitaxelgroup and the AZT group, where the AZT infusion was started one dayprior to the start of the paclitaxel injections. Animal weights andtumor sizes were measured on days 1, 3, 6, 8, and 10 after initiation ofthe paclitaxel treatment.

The antitumor effect of the drug treatments was measured in three ways.The first was the reduction in tumor size. Tumor sizes were determinedby first preparing a mold of the extruding tumor using Jeltrate, arapidly setting molding material, and then preparing and weighing thecountermold. Second, the apoptotic effect was measured. The animals wereeuthanized on day 10, and the tumors were harvested and fixed informalin. Histologic sections of 5 micron thickness were prepared andstained with hematoxylin and eosin. The tumor sections were evaluatedmorphologically for tumor cell density, and density of apoptotic cells.Because apoptotic cells disappear over time, the density ofnon-apoptotic cells is a secondary indicator of apoptosis. Celldensities were determined by counting the number of cells in fourrandomly selected microscopic fields at 400× magnification, using imageanalysis procedures (Au et al (2000) Proc Natl Acad Sci, In press).Third, the ability of drug treatment to prolong the survival time wasmeasured. For this study, the animals were monitored for 100 days, oruntil moribundity, defined by a tumor length exceeding 1.0 cm, wasreached.

The results, summarized in Table 4 and FIG. 9, showed that AZT enhancedthe in vivo antitumor effect of paclitaxel. First, treatment with thecombination of paclitaxel and AZT resulted in a decrease in tumor sizeduring a 10-day follow-up period, whereas animals in the control group,paclitaxel group, and AZT group showed an up to 4-fold increase in tumorsize. The tumor size of the animals receiving the combination ofpaclitaxel and AZT at 10 days was significantly smaller than all otherdose groups (p<0.001, ANOVA with repeated measures). Second, evaluationof the tumor morphology showed that the tumors of animals receiving thecombination of paclitaxel and AZT had a 2- to 4-fold higher density ofapoptotic cells, and a 2.6- to 4-fold lower density of non-apoptoticcells than all other dose groups (Table 4). Third, survival analysis(i.e., Kaplan Meier analysis) showed that the median time to reachmoribundity increased from 21-26 days for the control group and the AZTgroup to 42 days for the paclitaxel group and 49 days for thecombination group. The paclitaxel group did not show tumor-freesurvivors whereas the combination group showed 2 tumor-free survivors (2of 12, 16%). Survival of the group receiving the combination ofpaclitaxel and AZT was statistically longer than all other groups(p<0.01 by log rank test). The Kaplan-Meier curves for this study areshown in FIG. 8.

Treatments with single agents (either paclitaxel or AZT) producedminimal toxicity, with no toxicity-related death and minimal body weightloss compared to the pretreatment weight (<3%). The addition of AZT topaclitaxel did not enhance the body weight loss, indicating that AZT didnot enhance the host toxicity of paclitaxel. TABLE 4 Enhancement ofantitumor effect of paclitaxel by AZT. End-of- Number of Number ofexperiment nonapoptotic apoptotic % body weight, cells per cells perApoptotic % of 400× 400× cells per pretreatment Treatment (n) fieldfield tumor value Saline 235∀39  31∀7 12∀2% 106∀3 control (11) AZT (10)249∀ 36  28∀7 10∀3% 105∀6 Paclitaxel 168∀53 ^(a)  66∀34 ^(a) 30∀17% ^(a) 97∀6 ^(a) (12) paclitaxel +  64∀68 ^(b) 129∀36 ^(b) 72∀26% ^(b)  99∀4^(a) AZT (12)The average pretreatment body weights for the four groups ranged from 20g to 22 g. Data represents Mean″SD of four independent set ofexperiments. Cell density and apoptosis level were determined usingimage analysis at 4 randomized continuos tumor area per tumor.^(a) p < 0.05 compared to the control and AZT groups.^(b) p < 0.05 compared to all other groups.Equivalents

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, many equivalents to specificembodiments of the invention described specifically herein. Suchequivalents are intended to be encompassed in the scope of the followingclaims.

1-56. (canceled)
 57. A method for detecting telomerase activity in cellextract comprising: incubating a reaction mixture comprising a cellextract, a nucleic acid substrate for a telomerase, and nucleotidetriphosphates for a time sufficient for the nucleic acid substrate to bepolymerized; contacting the substrate with at least one nucleic acidprimer and subjecting the substrate to a polymerase chain reaction; anddetecting the presence of polymerase chain reaction products to detectthereby telomerase activity in said cell extract.
 58. The method ofclaim 57, wherein the cell extract is derived from a cell that has beencontacted with an agent.
 59. The method of claim 57, wherein the methodfurther comprises contacting the cell extract with an agent.
 60. Themethod of claim 57, wherein the agent is a telomerase inhibitory agent.61. The method of claim 60, wherein the telomerase inhibitory agent isAZT.
 62. The method of claim 60, wherein the telomerase inhibitory agentis d4T.
 63. The method of claim 57, wherein the telomerase inhibitoryagent is an antisense nucleic acid corresponding to a telomerase. 64.The method of claim 57, wherein the cell extract is derived from a humancell.
 65. The method of claim 59, wherein the nucleic acid substratecomprises the sequence provided in SEQ ID NO:
 10. 66. The method ofclaim 59, wherein the nucleic acid primer comprises the sequenceprovided in SEQ ID NOS: 1 and
 2. 67. The method of claim 59 wherein thenucleic acid primer is labeled with a radioisotope.
 68. The method ofclaim 59 wherein said nucleic acid primer is labeled with a fluorescentlabel.
 69. A method for determining telomere length comprising:hybridizing telomeric DNA fragments with a telomere probe; anddetermining the amount of hybridized telomere probe present, whereby theamount of hybridized telomere probe present is an indication of telomerelength.
 70. The method of claim 69, wherein the telomeric DNA fragmentsare produced using a restriction enzyme.
 71. The method of claim 70,wherein the restriction enzyme or enzymes is selected from the groupconsisting of HinfI, HaeIII, and HhaI.
 72. The method of claim 69,wherein the telomeric DNA is derived from a cell.
 73. The method ofclaim 69, wherein the cell has been contacted with an agent.
 74. Themethod of claim 73, wherein the agent is a telomerase inhibitory agent.75. The method of claim 74, wherein the telomerase inhibitory agent isAZT.
 76. The method of claim 74, wherein the telomerase inhibitory agentis d4T.
 77. The method of claim 74, wherein the telomerase inhibitoryagent is an antisense nucleic acid corresponding to a telomerase. 78.The method of claim 72, wherein the cell is from a human.
 79. The methodof claim 69, wherein the telomere probe comprises the sequence providedin SEQ ID NO:
 10. 80. The method of claim 69, wherein the telomere probecomprises the sequence provided in SEQ ID NO:
 11. 81. The method ofclaim 69, wherein the telomere probe is labeled with a radioisotope. 82.The method of claim 69, wherein the telomere probe is labeled with afluorescent label.
 83. The method of claim 1, wherein said telomeredamage inducing agent is formulated as a nanoparticle comprising a crosslinked gelatin.
 84. The method of claim 1, wherein said telomeraseinhibitory agent is formulated as a nanoparticle comprising a crosslinked gelatin.
 85. The method of any one of claims 83 or 84, whereinsaid nanoparticle is about 500 nm to about 1 μm in diameter.
 86. Themethod of claim 1, wherein said telomere damage inducing agent isformulated as a microparticle.
 87. The method of claim 1, wherein saidtelomerase inhibitory agent is formulated as a microparticle.
 88. Themethod of any one of claims 86 or 87, wherein said microparticle isabout 1 μm to about 10 μm in diameter.
 89. (canceled)